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Biochemistry

Laboratory Manual

DR.GYANENDRA AWASTHIDR.GYANENDRA AWASTHIDR.GYANENDRA AWASTHIDR.GYANENDRA AWASTHI

DR.SANTOSH KUMARDR.SANTOSH KUMARDR.SANTOSH KUMARDR.SANTOSH KUMAR

DR.ASHWANI SANGHIDR.ASHWANI SANGHIDR.ASHWANI SANGHIDR.ASHWANI SANGHI

MR.SHIV SHARAN SINGHMR.SHIV SHARAN SINGHMR.SHIV SHARAN SINGHMR.SHIV SHARAN SINGH

2013

International E – Publication

www.isca.me , www.isca.co.in

BIOCHEMISTRY LABORATORY MANUALBIOCHEMISTRY LABORATORY MANUALBIOCHEMISTRY LABORATORY MANUALBIOCHEMISTRY LABORATORY MANUAL

DR.GYANENDRA AWASTHIDR.GYANENDRA AWASTHIDR.GYANENDRA AWASTHIDR.GYANENDRA AWASTHI

DR.SANTOSH KUMARDR.SANTOSH KUMARDR.SANTOSH KUMARDR.SANTOSH KUMAR

DR.ASHWANI SANGHIDR.ASHWANI SANGHIDR.ASHWANI SANGHIDR.ASHWANI SANGHI

MR.SHIV SHARAN SINGHMR.SHIV SHARAN SINGHMR.SHIV SHARAN SINGHMR.SHIV SHARAN SINGH

Department of Biochemistry

Dolphin (PG) Institute of Biomedical & Natural Sciences,

DEHRA DUN (UTTARAKHAND)

2013

International E - Publication


International E - Publication 427, Palhar Nagar, RAPTC, VIP-Road, Indore-452005 (MP) INDIA

Phone: +91-731-2616100, Mobile: +91-80570-83382

E-mail: [email protected] , Website: www.isca.me , www.isca.co.in

© Copyright Reserved

2013

All rights reserved. No part of this publication may be reproduced, stored, in a

retrieval system or transmitted, in any form or by any means, electronic,

mechanical, photocopying, reordering or otherwise, without the prior permission

of the publisher.

ISBN: 978-93-83520-17-6



International Science Congress Association

iii

Author’s Preface

Living systems are shaped by an enormous variety of biochemical reactions which can be

understand via various biochemical techniques. In the present Manual an effort has been made to

discuss these biochemical techniques in simple and lucid manner so that reader can have

comprehensive understanding of the subject. Unlike other basic science subjects like Chemistry,

Zoology and Botany, Biochemistry practical’s generally require a variety of chemicals and

expensive equipments.

One of the highlight of the present manual is that it covers the practical aspects of different

biochemical techniques for undergraduate and postgraduate students of life sciences. The manual

is divided into seven main sections, each of which subdivided into chapters. First section deals

with buffers, pH and solution preparation mainly. Second and third unit deals with analysis of

biomolecules both qualitatively and quantitatively. The fourth, fifth and sixth unit mainly

concerned with chromatographic, electrophoretic and spectroscopic techniques. The last unit is

regarding demonstration of PCR and ELISA.

The present script is just a compilation of facts and interpretation from different sources. The

Authors does not claim the originality of the subjects. The present manual is the author’s

understandings of the various techniques described and are fully responsible for the errors and

misinterpretations.

Dr.Gyanendra Awasthi Dr.Santosh Kumar

Head & Reader, Assistant Professor,

Department of Biochemistry, Department of Biochemistry,

DIBNS, Dehradun DIBNS, Dehradun

Dr.Ashwani Sanghi Mr.Shiv Sharan Singh

Assistant Professor, Assistant Professor,

Department of Biochemistry, Department of Biochemistry,

DIBNS, Dehradun DIBNS, Dehradun




iv

INDEX

Exp.No. Name Of The Experiment Page No.

Section I: Solutions, Buffers & pH

01. Solution 2 – 3

02. Buffers 4 – 9

03. pH 10 – 11

Section II: Qualitative Analysis Of Biomolecules

04. Molisch’s Test 13 – 14

05. Iodine Test 15 – 16

06. Benedict’s’s Test 17 – 18

07. Barfoed’s Test 19 – 20

08. Seliwanoff’s Test 21 – 22

09. Bial’s Test 23 – 24

10. Biuret Test 28 – 29

11. Ninhydrin Test 30 – 32

12. Xanthoproteic Test 33 – 34

13. Millon’s Test 35 – 36

14. Sakaguchi’s Test 37 – 38

15. Lipids Solubility Test 41

16. Acrolein Test 42

17. Zak Test 43

Section III: Quantitative Analysis Of Biomolecules

18. Ferricyanide Assay 45 – 46

19. Lowry’s Assay 47 – 49

20. Acid Value Determination 50 – 51

21. Saponification Value Determination 52 – 55

Section IV: Chromatographic Techniques

22. Ascending Paper Chromatography 58 – 62

23. Thin Layer Chromatography 63 – 66

Section V: Electrophoretic Techniques

24. Agarose Gel Electrophoresis 69 – 70

25. PAGE 71 – 75

Section VI: Spectroscopic Techniques

26. (a.) Verification of Beer’s Law 77 – 80

26. (b.) Determinmation of �max

Section VII: Laboratory Demonstrations

27. Polymerase Chain Reaction 82 – 86

28. ELISA 87 - 91

ABOUT AUTHOR 92




1

Section: I

Solutions, Buffers & pHSolutions, Buffers & pHSolutions, Buffers & pHSolutions, Buffers & pH




2

Experiment No. 01

AIM: Preparation Of Normal, Molar & Percent Solutions.

Molarity (M) :

This is the most common method for expressing the concentration of a solution in

biochemical studies. The molarity of a solution is the number of moles of the

solute dissolved per L of the solution. A solution which contains 1 mole of the

solute in one L of the solution is called a molar solution. Molarity of a solution can

be calculated as follows:

Weight of a solute in g/L of solution

Molarity =

Mol. Wt. of solute

It may be noted that in case of molar solutions, the combined total volume of the

solute and solvent is one L. Thus for preparing 0.1 M NaOH, one may proceed as

follows:

Mol. Wt. of NaOH = 40

Required molarity of solution = 0.1M

Amount (in g) of NaOH per L of solution = Mol. Wt.of NaOH x molarity

= 40 x 0.1= 4 g

Thus, weigh 4 g of NaOH, dissolve it in a small volume of solvent (water) and

make the final volume to 1 L with the solvent.

Sometime it is desirable to know number of moles of a substance in a reaction

mixture. This can be calculated using a simple relationship:

1 M solution = 1 mole of the substance/L of solution.

= 1 mmole/ml of solution

= 1 µmole/µl of solution

1 mM solution = 1 mmole/L of solution




3

= 1 µmole/ml of solution

Normality (N):

The normality of a solution is the number of gram equivalents of the solute per L

of the solution. Therefore,

Amount of a substance in g/L of solution

Normality =

Eq. wt. of substance

For preparing 0.1 N Na2CO3 (Eq.wt. of Na2CO3= 53) solution, dissolve 5.3g Na2CO3

in a final volume of 1 L of solution.

Percentage by Mass or % (w/w):

It is the weight of the component present in 100 parts by weight of the solution.

In a solution containing 10g sugar in 40g of water, then

10x100

Mass % of sugar = = 20%

(10+40)

Percentage by volume or % (v/v) :

It is the volume of the component in 100 parts by volume of the solution. In a

solution containing 20 ml alcohol in 80 ml of water, the % volume of alcohol will

be

20 x 100 = 20%

(20 + 80)




4

EXPERIMENT NO.02

AIM: To Prepare Buffer Solutions.

PHOSPHATE BUFFER: Phosphate salts are known by several names and the

correct phosphate must be used to prepare buffer solutions. One phosphate

cannot be substituted for another phosphate. Check formula of salt to be certain.

Formula Name of salt Other names

KH2PO4 potassium dihydrogen phosphate

potassium dihydrogen orthophosphate

monobasic potassium phosphate

monopotassium phosphate

acid potassium phosphate

potassium biphosphate

K2HPO4 potassium hydrogen phosphate

dipotassium hydrogen orthophosphate

dipotassium hydrogen phosphate

dibasic potassium phosphate

dipotassium phosphate

K3PO4 potassium phosphate tribasic potassium phosphate

tripotassium phosphate

Standardization buffers (For pH=7.00): Add 29.1 ml of 0.1 molar NaOH to 50 ml

0.1 molar potassium dihydrogen phosphate. Alternatively: Dissolve 1.20g of

sodium dihydrogen phosphate and 0.885g of disidium hydrogen phosphate in 1

liter volume distilled water.

Standardization buffers (For pH= 4.00): Add 0.1 ml of 0.1 molar NaOH to 50 ml of

0.1 molar potassium hydrogen phthalate. Alternatively, Dissolve 8.954g of

disodium hydrogen phosphste.12 H2O and 3.4023g of potassium di hydrogen

phosphate in 1 liter volume distilled water.

RANGE OF COMMON BUFFER SYSTEMS:

Buffering System Buffering pH

Range @ 25°C

Hydrochloric acid/ Potassium chloride 1.0 - 2.2




5

Glycine/ Hydrochloric acid 2.2 - 3.6

Potassium hydrogen phthalate/ Hydrochloric acid 2.2 - 4.0

Citric acid/ Sodium citrate 3.0 - 6.2

Sodium acetate/ Acetic acid 3.7 - 5.6

Potassium hydrogen phtaalate/ Sodium hydroxide 4.1 - 5.9

Disodium hydrogen phthalate / Sodium dihydrogen orthophospate 5.8 - 8.0

Dipotassium hydrogen phthalate / Potassium dihydrogen orthophospate 5.8 - 8.0

Potassium dihydrogen orthophosphate / sodium hydroxide 5.8 - 8.0

Barbitone sodium / Hydrochloric acid 6.8 - 9.6

Tris (hydroxylmethyl) aminomethane / Hydrochloric acid 7.0 - 9.0

Sodium tetraborate/ Hydrochloric acid 8.1 - 9.2

Glycine/ Sodium hydroxide 8.6 - 10.6

Sodium carbonate/ Sodium hydrogen carbonate 9.2 - 10.8

Sodium tetraborate/ Sodium hydroxide 9.3 - 10.7

Sodium bicarbonate / Sodium hydroxide 9.60 - 11.0

Sodium hydrogen orthophosphate / Sodium hydroxide 11.0 - 11.9

Potassium chloride/ Sodium hydroxide 12.0 - 13.0

PREPARING A BUFFER SOLUTION: This page gives tabulated info on the

preparation of buffers by mixing adjusters with a known volume of the primary

salt solution, and made up to 200 ml with distilled water.

BUFFERS (pH: 1- 9)

Buffer A :

pH 1.0 - 2.2

Buffer B :

pH 2.2 - 4.00

Buffer C :

pH 4.10 - 5.90

Buffer D :

pH 5.8 - 8.00

Buffer E :

pH 7.0 - 9.00

50 ml 0.2 M KCl

+ ml of 0.2 M

HCl

100 ml 0.1 M

potassium

hydrogen

phthalate + ml

of 0.1 M HCl

100 ml 0.1 M

potassium

hydrogen

phthalate + ml

of 0.1 M NaOH

100 ml 0.1 M

KH2PO4 + ml of

0.1 M NaOH

100 ml 0.1 M tris

(hydroxymethyl)

aminomethane +

ml of 0.1 M HCl




6

pH ml of 0.2M HCl

added pH

ml of 0.1M HCl

added pH

1.00 134.0 2.20 99.0 4.10

1.10 105.6 2.30 91.6 4.20

1.20 85.0 2.40 84.4 4.30

1.30 67.2 2.50 77.6 4.40

1.40 53.2 2.60 70.8 4.50

1.50 41.4 2.70 64.2 4.60

1.60 32.4 2.80 57.8 4.70

1.70 26.0 2.90 51.4 4.80

1.80 20.4 3.00 44.6 4.90

1.90 16.2 3.10 37.6 5.00

2.00 13.0 3.20 31.4 5.10

2.10 10.2 3.30 25.8 5.20

2.20 7.8 3.40 20.8 5.30

3.50 16.4 5.40

3.60 12.6 5.50

3.70 9.0 5.60

3.80 5.8 5.70

3.90 2.8 5.80

4.00 0.2 5.90




7

BUFFERS (pH: 8 – 13)

Buffer F:

pH 8.0 - 9.10

Buffer G :

pH 9.2 - 10.80

Buffer H :

pH 9.60 - 11.00

Buffer I :

pH 10.90 - 12.00

Buffer J :

pH 12.00 - 13.00

100 ml 0.025 M

Na2B4O7.10H2O

(borax) + ml of

0.1 M HCl

100 ml 0.025 M

Na2B4O7.10H2O

(borax) + ml of

0.1 M NaOH

100 ml 0.05 M

NaHCO3 + ml of

0.1 M NaOH

100 ml 0.05 M

Na2HPO4 + ml of

0.1 M NaOH

50 ml 0.2 M KCl

+ volume

indicated (in ml)

0.2 M NaOH

pH ml of 0.1M HCl

added pH

ml of 0.1M

NaOH added pH

8.00 41.0 9.20 1.8 9.60

8.10 39.4 9.30 7.2 9.70

8.20 37.6 9.40 12.4 9.80

8.30 35.4 9.50 17.6 9.90

8.40 33.2 9.60 22.2 10.00

8.50 30.4 9.70 26.2 10.10

8.60 27.0 9.80 30.0 10.20

8.70 23.2 9.90 33.4 10.30

8.80 19.2 10.00 36.6 10.40

8.90 14.2 10.10 39.0 10.50

9.00 9.2 10.20 41.0 10.60

9.10 4.0 10.30 42.6 10.70

10.40 44.2 10.80

10.50 45.4 10.90

10.60 46.6 11.00

10.70 47.6

10.80 48.5




8

ACETATE BUFFER SOLUTIONS (pH 3 – 6): Make up the following solutions-

(1) 0.1M acetic acid

(2) 0.1M sodium acetate (tri-hydrate) (13.6 g/L )

Mix in the following proportions to get the required pH

pH vol. of 0.1M

acetic acid

vol. of 0.1M

sodium acetate

3 982.3 ml 17.7 ml

4 847.0 ml 153.0 ml

5 357.0 ml 643.0 ml

6 52.2 ml 947.8 ml

PHOSPHATE BUFFER SOLUTIONS (pH 7 – 11): Make up the following solutions-

(1) 0.1M disodium hydrogen phosphate (14.2g /L)

(2) 0.1M HCl

(3) 0.1M NaOH

Mix in the following proportions to get the required pH

pH vol. of

phosphate

vol. of 0.1M

HCl

vol. of 0.1M

NaOH

7 756.0 ml 244 ml

8 955.1 ml 44.9 ml

9 955.0 ml 45.0 ml

10 966.4 ml 33.6

11 965.3 ml 34.7

Addition of acid or base to a salt (pH 3 – 11)

Here, the primary salt is a solid and is weighed out in grams. A measured amount

of 0.1M HCl or NaOH is added, then made up to 1 liter to give the relevant buffer

solution.




9

pH Salt mixture

Dilute each mixture to 1 liter solution with distilled water

3 10.21g potassium hydrogen phthalate and 223 ml of 0.10M HCl

4 10.21g potassium hydrogen phthalate and 1ml of 0.10M HCl

5 10.21g potassium hydrogen phthalate and 226ml of 0.10M NaOH

6 6.81g potassium dihydrogen phOsphate and 56ml of 0.10M NaOH

7 6.81g potassium dihydrogen phosphate and 291ml of 0.10M NaOH

8 6.81g potassium dihydrogen phosphate and 467ml of 0.10M NaOH

9 4.77g sodium tetraborate and 46ml of 0.10M HCl

10 4.77g sodium tetraborate and 183ml of 0.10M NaOH

11 2.10g sodium bicarbonate and 227ml of 0.10M NaOH

McIlvaine’s buffer (pH 7.20): 173.9 ml of 0.2 M Na2HPO4 and 26.1 ml of 0.1 M

citric acid were mixed to prepare the buffer of pH 7.2 and the final pH adjustment

was done by addition of either of the two solutions simultaneously.




10

Experiment No.03

AIM: To Find Out The Strength Of The Given Hydrochloric Acid Solution (Approx.

Strength N/10) By Titrating It Against Sodium Hydroxide Using pH Meter.

APPARATUS: pH meter with glass electrode, reference electrode, beaker, burette,

stirrer etc.

CHEMICALS: HCl, NaOH.

THEORY: When an alkali is added to an acid solution, the pH of the solution

increases slowly. But at the equivalence point, the rate of change of the solution

is very rapid. A plot is drawn between volume of the alkali added and the pH of

the solution. The sharp break in the curve gives the equivalence point, from which

the strength can be calculated using normality equation.

INSTRUMENTATION: In pH meter the glass electrode is incorporated in an

ordinary potentiometric circuit. The potentiometric pH meter differs from a simple

potentiometer to the extent that the galvanometer is replaced by an electronic

circuit that amplifies the current in the cell circuit by a factor of 109 or more.

Before using, the pH meter is first standardised with a buffer solution of known p

H.

Then the glass and reference electrode are immersed in an unknown solution and

the pH is read directly on p

H scale.

PROCEDURE:

1) Caliberate the pH meter with the glass electrode in the buffer solution of

known pH.

2) Wash the glass electrode and the reference electrode with distilled water

and then rinse with the acid solution.

3) Take 5ml of HCl solution in a beaker. Add sufficient water so as reference

and glass electrodes are completely dipped.

4) Note down the pH of the pure acid solution.

5) Now add 10ml of N/10 NaOH from the burette and note down the pH after

each addition.




11

6) Continue adding NaOH solution from the burette and note down the pH

after each addition.

7) Near the equivalence point the change in pH is much more rapid than in any

other region.

OBSERVATION:Volume of acid taken = 5ml

Vol. of alkali added 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0

CALCULATION: Plot a curve with pH

values as ordinate and volume of alkali added

as abscissa. The sharp break in curve corresponds to the equivalence point.

Volume of alkali added (ml)

Let the volume of alkali at equivalence point = x mL

Acid alkali

N1V1 = N2V2

N1 X 5 = N/10 X x

N1 = N/10 X

Strength of HCl solution

= 36.5 X x/ 10 X 5 g/L

RESULT: The strength of given acid solution is ……. g/l

PRECAUTIONS:

1) The pH

meter should be caliberated before use.

2) After addition of alkali, the solution should be thoroughly stirred.

3) Electrodes must be immersed properly in the solution.




12

Section: II

Qualitative Analysis Of Qualitative Analysis Of Qualitative Analysis Of Qualitative Analysis Of

BiomoleculesBiomoleculesBiomoleculesBiomolecules




13

Experiment No.04

AIM: To Detect The Presence Of Carbohydrate In The Given Samples By

Molisch’s Test.

PRINCIPLE: Molisch's test (named after Austrian botanist Hans Molisch) is a

sensitive chemical test for the presence of carbohydrates, based on the

dehydration of the carbohydrate by sulfuric acid to produce an aldehyde, which

condenses with two molecules of phenol (usually α-naphthol, though other

phenols (e.g. resorcinol, thymol) also give colored products), resulting in a red- or

purple-colored compound.

All carbohydrates (larger than tetroses) – monosaccharides, disaccharides, and

polysaccharides – should give a positive reaction, and nucleic acids and

glycoproteins also give a positive reaction, as all these compounds are eventually

hydrolyzed to monosaccharides by strong mineral acids. Pentoses are then

dehydrated to furfural, while hexoses are dehydrated to 5-hydroxymethylfurfural.

Either of these aldehydes, if present, will condense with two molecules of

naphthol to form a purple-colored product, as illustrated below by the example of

glucose:

REAGENTS:




14

1) 5% α-napthol in 95% alcohol

2) Concentrated H2SO4

3) 1% solution different carbohydrates.

PROCEDURE: Add 2-3 drops of α- naphthol solution to 2ml of test solution. Very

gently pipette 1ml conc. H2SO4 along the side of the test tube so that the 2

distinct layers are formed. Carefully observe any colour change at the junction of

2 layers. Appearance of purple colour indicates the presence of carbohydrates in

the sample preparation or the test solution.

OBSERVATION TABLE:

Si.No. Sample Initial Observation Final Observation Interpretation

1. 1% Glucose

2. 1% Fructose

3. 1% Ribose

4. 1% Maltose

5. 1% Sucrose

6. 1% Starch

7. 1% Glycogen

8. Water

RESULT & CONCLUSION:




15

Experiment No.05

AIM: To Detect The Presence Of Polysaccharides (Starch & Glycogen) In The

Given Samples By Iodine Test.

PRINCIPLE: The Iodine test is used to test for the presence of starch. Iodine

solution — iodine dissolved in an aqueous solution of potassium iodide — reacts

with the starch producing a purple black/blue black color.

Iodine forms coloured adsorption complexes with polysaccharides. Starch gives

blue colour with iodine, while glycogen gives reddish brown coloured complex.

Hence it is a useful, convenient method for the detection of amylase, amylopectin

& glycogen.




16

REAGENTS:

1) Iodine solution: prepare 0.005N iodine solution in 3% (w/v) potassium iodide

solution.

2) Sugar solution: 1% solution of different carbohydrates.

PROCEDURE: 1.0 ml of test solution in a test tube & added a drop of iodine

solution in each test tube. A blank is performed with water. Test tube is shaken

and color is observed. Test tube in which color is developed is heated & change in

color observed now test tube is cooled & change in color observed.

OBSERVATION TABLE:


1. 1% Glucose

2. 1% Fructose

3. 1% Ribose

4. 1% Maltose

5. 1% Sucrose

6. 1% Starch

7. 1% Glycogen

8. Water





17

Experiment No.06

AIM: To Detect The Presence Of Reducing Sugar In The Given Samples By

Benedict’s Test.

PRINCIPLE: Carbohydrates with free or potentially free reducing groups easily

reduce metal like copper (Cu), Ba, Hg (mercury), Iron (Fe) & silver (Ag) in Alkaline

solution when blue alkaline cupric oxide or hydroxide suspended in alkaline

medium is heated it forms blue precipitate of cupric oxide (CuO ) but in presence

of reducing substances, e.g reducing sugars having free or potentially free

aldehyde or ketonic group upon heating blue cupric hydroxide converted into

insoluble brownish red cuprous oxide (Cu2O) suspensions of metal hydroxide,

used in metal reduction test and to precipitate in alkaline medium to check that

organic compound having more than one alcoholic groups are added to give free

metals. This test is more sensitive and reagent does not deteriorate if stored for a

longer time. In this method the sodium citrate functions as a chelating agent by

forming soluble complex ions with Cu++, preventing the precipitation of CuCO3

in alkaline solutions. Presence of reducing sugar results in the formation of red

precipitate of cuprous oxide. Depending on the concentration of sugars, yellow to

green color is developed. All monosaccharides are reducing sugars as they all

have a free reactive carbonyl group. Some disaccharides like maltose have

exposed carbonyl groups and are also reducing sugars but less reactive than

monosaccharides.

D-glucose + 2CuO D-gluconic acid + Cu2O

(Blue) (Brick red precipitate)

REAGENTS:




18

1) Benedict’s reagent A: Dissolve 173 gm of sodium citrate & 100gm of anhydrous

Na2CO3 in 600ml of hot H2O. Dilute to the 800ml with water.

2) Benedict’s reagent B: Dissolve 17.3g of CuSO4.5H2O in 100ml hot water. Cool &

l % dilute to 100ml.

Add both reagents with constant stirring. Make the final volume to 1L.


PROCEDURE: Add 0.5-1ml of the test solution or sample extract to 5ml of

Benedict’s reagent. Keep the test tubes in vigorously heated boiling water bath.

Cool the solution. Observe the colour change from blue to green, yellow, orange

or red depending upon the amount of reducing sugar present in the test sample.

OBSERVATION TABLE:


1. 1% Glucose

2. 1% Fructose

3. 1% Ribose

4. 1% Maltose

5. 1% Sucrose

6. 1% Starch

7. 1% Glycogen

8. Water





19

Experiment No.07

AIM: To Differentiate Between Monosaccharides & Reducing Disaccharides By

Barfoed’s Test.

PRINCIPLE: Barfoed's Test is a chemical test used for detecting the presence of

monosaccharides. It was invented by Danish chemist Christen Thomsen Barfoed

and is primarily used in botany. The test is similar to the reaction of Benedict's

solution to aldehydes, except that reduction of copper occurs in acidic medium

rather alkaline medium.

Barfoed's reagent, a mixture of ethanoic (acetic) acid and copper(II) acetate, is

combined with the test solution and boiled. A red copper(II) oxide precipitate is

formed will indicates the presence of reducing sugar. The reaction will be

negative in the presence of disaccharide sugars because they are weaker reducing

agents. This test is specific for monosaccharides . Due to the weakly acidic nature

of Barfoed's reagent, it is reduced only by monosaccharides.

Disaccharides may also react, but the reaction is much slower.The aldehyde

group of the monosaccharide which normally forms a cyclic hemiacetal is

oxidized to the carboxylate. Monosaccharides usually react in about 1-2min while

the reducing disaccharides take much longer time between 7-12min to get

hydrolysed & then react with the reagent.

D-glucose + 2CuO D-gluconic acid + Cu2O

REAGENTS:

1) Barfoed’s regent: Dissolve 13.3g of copper acetate in 200ml water & 1.8 ml of

glacial acetic acid to it.





20

PROCEDURE: 1.0 ml of test solution was taken and to it added 2 ml of Barfoed’s

regent and it was then boiled for 1-2 min and allowed to stand for few minutes.

OBSERVATION TABLE:


1. 1% Glucose

2. 1% Fructose

3. 1% Ribose

4. 1% Maltose

5. 1% Sucrose

6. 1% Starch

7. 1% Glycogen

8. Water


COMMENTS:

1) This test is not specific for glucose or any other monosaccharides but simply

used to detect reducing sugars.

2) Disaccharides also respond to this test.

3) This test is copper reduction test but it differs from Fehling’s or Benedict’s test

in that reduction is brought about in acid solution.

4) Chloride interferes in this test and therefore unsuitable for detection of sugar

in urine or any other body fluid containing Cl.




21

Experiment No.08

AIM: To Detect The Presence Of Ketose Sugars In The Given Samples By

Seliwanoff’s Test.

PRINCIPLE: It is a color reaction specific for ketoses. One can distinguish aldoses

from ketoses based on their ability to form furfurals. When conc. HCl is added,

ketoses undergo dehydration to yield furfural derivatives more rapidly than

aldoses. These derivatives form complexes with resorcinol to yield deep red color.

The test reagent causes the dehydration of ketohexoses to form 5-

hydroxymethylfurfural. 5-hydroxymethylfurfural reacts with resorcinol present in

the test reagent to produce a red product within two minutes. Aldohexoses

reacts so more slowly to form the same product. Aldoses generally exist in

solution as pyranoses, whereas ketoses generally exist as furanoses, hence the

ability of ketoses to rapidly dehydrate to yield furfurals:

REAGENTS: A




22

1) Seliwanoff’s reagent- 0.05% (w/v) resorcinol in 3 N HCl.

2) 1% solution of different carbohydrates.

PROCEDURE: 2.0 ml of seliwonoff’s reagent was taken in a test tube and 0.5 ml of

test solution was added to this. Test tube was placed in boiling water bath. Test

was performed with different carbohydrates and with water as blank. A cherry

red condensation product will be observed indicating the presence of ketoses in

the test sample. There will be no significant change in colour produced for aldose

sugar.

OBSERVATION TABLE:


1. 1% Glucose

2. 1% Fructose

3. 1% Ribose

4. 1% Maltose

5. 1% Sucrose

6. 1% Starch

7. 1% Glycogen

8. Water


COMMENT: Prolonged heating will hydrolyse polysaccharides and may interfere

in this test.




23

Experiment No.09

AIM: To Detect The Presence Of Pentose Sugar In The Given Samples By Bial’s

Test.

PRINCIPLE: Bial’s test can be used to distinguish pentoses from hexoses. In the

presence of concentrated HCl, pentoses react to give furfural, whereas hexoses

give hydroxymethyfurfural. Orcinol and furfural condense in the presence of ferric

ion to form a colored product. Appearance of green colour or precipitate indicates

the presence of. Hexoses, which give 5-hydroxyfurfural on dehydration, react with

Bial’s reagent to give a brownish colour. Di- and polysaccharides give the same

results but at a much slower rate:

REAGENTS:

1) Bial’s reagent: Dissolve 1.5 gm of orcinol in 100ml of conc. HCl & add 20-30

drops of 10% ferric chloride solution to it.

2) 1% solution of different carbohydrates.




24

PROCEDURE: 1.0 ml of sugar solution added to about 2.0ml of bial’s reagent &

heated until boiling, a blue green color indicates presence of a pentose sugar. Test

is performed with different carbohydrates and water as blank.

OBSERVATION TABLE:


1. 1% Glucose

2. 1% Fructose

3. 1% Ribose

4. 1% Maltose

5. 1% Sucrose

6. 1% Starch

7. 1% Glycogen

8. Water





25

Summary: Carbohydrates Qualitative Analysis

No. Test Observation Inference Reaction

1

Molisch’s Test

2-3 drops of beta-

naphthol solution is

added to 2ml of the test

solution. Very gently

added 1ml of Conc.

H2SO4 along the side of

the test tube.

A deep violet

coloration is

produced at the

junction of two

layers.

Presence of

carbohydrates.

This is due to the

formation of an

unstable condensation

product of beta-

naphthol with furfural

(produced by the

dehydration of the

carbohydrate)

2

Iodine test

4-5 drops of iodine

solution is added to 1ml

of the test solution and

mixed the contents

gently

Blue colour is

observed

Presence of

polysaccharide

Iodine forms coloured

adsorption complexes

with polysaccharides

3

Benedict’s test

To 5 ml of Benedict's

solution, add 1ml of the

test solution and shake

each tube. Place the tube

in a boiling water bath

and heat for 3 minutes.

Remove the tubes from

the heat and allow them

to cool.

Formation of a green,

red, or yellow

precipitate

Presence of

reducing sugars

If the saccharide is a

reducing sugar it will

reduce Copper [Cu]

(11) ions to Cu(1)

oxide, a red precipitate

5

Barfoed’s test

To 2 ml of the solution to

be tested added 2 ml of

freshly prepared

Barfoed's reagent. Place

test tubes into a boiling

water bath and heat for 3

minutes. Allow to cool.

A deep blue colour is

formed with a red

ppt. settling down at

the bottom or sides

of the test tube.

Presence of

reducing sugars

[appearance of a

red ppt as a thin

film at the bottom

of the test tube

within 3-5 min. is

indicative of

reducing mono-

saccharide. If the

ppt formation takes

If the saccharide is a

reducing sugar it will

reduce Cu (11) ions to

Cu(1) oxide




26

more time then it is

a reducing

disaccharide

6

Seliwanoff test

To 3ml of of Seliwanoff’s

reagent, add 1ml of the

test solution, boil in

water bath for 2 minutes

A cherry red colored

precipitate within 5

minute is obtained

A faint red colour

produced

Presence of ketoses

[Sucrose gives a

positive ketohexose

test ]

Presence of aldoses

When reacted with

Seliwanoff reagent,

ketoses react within 2

minutes forming a

cherry red

condensation product

Aldopentoses react

slowly forming the

coloured condensation

product

7

Bials test

Add 3ml of Bial’s reagent

to 0.2ml of the test

solution. heat the

solution in a boiling

water bath for 2 minutes

A blue-green product

A muddy brown to

gray product

Presence of

pentoses

Presence of

hexoses,

The furfurals formed

produces condensation

products with specific

colour




27

Differences Encountered In A Real Laboratory

In an actual laboratory setting, there are certain important steps that are not

necessarily applicable in a virtual lab:

1. Always wear lab coat and gloves when you are in the lab. When you enter the

lab,switch on the exhaust fan and make sure that all the reagents required for

the experiment are available. If it is not available, prepare the reagents using

the components for reagent preparation.

2. Care should be taken while handling caustic acids like Conc. Sulphuric acid

[H2SO4], nitric acid [HNO3], Hydrochloric acid [HCl]. These acids should be

opened and used in FUMEHOOD only. Accidental spill of these acids will cause

severe burns and itching. Wash the spilled area with cold water and inform the

lab assistant immediately.

3. When Sodium hydroxide is prepared, make sure that it is handled with care as

the sodium hydroxide solution is caustic in nature.

4. Always check the water level in the water bath and if it is up to the level [nearly

half the volume], switch on the water bath and adjust to the required

temperature. Take care while using the water bath for the boiling step in the

experiment. Hold the test tube using a test tube holder.

5. There should be a proportion between the reagents added and the test

solution to obtain good result within the time mentioned. The droppers used

should not be mixed between the reagents, always use individual droppers for

each reagent.

6. The color formed will depend upon the quality of the reagents. So care should

be taken while preparing the reagents. If commercially available reagents are

used assure that it is not kept open for long time.

7. Clean the test tubes and glass wares with soap and distilled water. Recap the

reagent bottles once the experiment is completed. The water bath and the

exhaust fan should be switched off.




28

Experiment No.10

AIM: To Detect The Presence Of Peptide Bonds In The Given Samples By Biuret

Test.

PRINCIPLE: The biuret test will indicate the presence of amino acid residues of

peptides containing two or more amino acid residues and therefore is used to

determine whether or not a protein is present. This test relies on the fact that

amino acid residues form a colored complex with Cu+2

ion in basic medium:

The test is given by those substances which contain at least two carbonyl group

joined either directly through a single atom of carbon or nitrogen. In this test

alkaline CuSO4 reacts with compounds containing two or more peptide bond

giving a violet colored complex. This biuret test is apparently due to co-ordination

of cupric- ion with the unshared electron pair of peptide nitrogen and oxygen of

water to form coloured co-ordination complex which may be represented. All

proteins should give a positive test whereas simple amino acids should give a

negative test.

REAGENTS:

1) 1% CuSO4.5H2O solution

2) 40% NaOH

3) 0.5% protein- solution of bovine serum albumin & casein in NaOH

4) 0.5% amino acid solution

PROCEDURE: 1ml of sample solution was taken in a test tube & 0.5ml of NaOH is

added & mix well. 2-5 drops of CuSO4 solution was added. Observe for the pink or

violet colour shows presence of peptides or proteins in the sample.




29

OBSERVATION TABLE:


1. 0.5% Glycine

2. 0.5% BSA

3. 0.5% Casein

4. 0.5% Urea


COMMENTS:

1) Dipeptides do not give this test. Two or more peptide linkages being

required.

2) Presence of MgSO4 in solution to be tested interfere with reaction because

of precipitation of Mg(OH)2.




30

Experiment No.11

AIM: To Detect The Presence Of Amino Acids In The Given Samples By Ninhydrin

Test.

PRINCIPLE: This is due to a reaction between amino group of free amino acid and

ninhydrin (triketohydrindene hydrate). Ninhydrin is a powerful oxidizing agent

and in its presence, amino acid undergo oxidative determination liberating

ammonia, CO2, a corresponding aldehyde and reduced form of ninhydrin. The

ammonia formed from amino group react with ninhydrin and its reduced product

(hydridantin) to give a blue substrate diketohydrin (ruhemann’s purple) however,

in case of imino acid like proline, a different product having a bright yellow colour

is formed. Asparagine which has a free amide group reacts to give a brown

coloured product. This test is also given by protein and peptides.

Ruhemann’s Purple




31

REAGENTS:

1) Boiling water bath.

2) Ninhydrin: 0.2% solution prepared in acetone.

3) Test solution: prepare solutions containing 0.5% of different amino acids.

PROCEDURE: Add 2-5 drop of ninhydrin solution to 1ml of test solution or sample

preparation mix and keep for 5min in boiling water bath and observe the

development of pink, purple or violet-blue colour. Imino acid like proline and

hydroxyproline give a yellow coloured complex.




32

OBSERVATION TABLE:


1. 0.5% Glycine

2. 0.5% BSA

3. 0.5% Proline

4. 0.5% Asparagine





33

Experiment No.12

AIM: To Detect The Presence Of Aromatic Amino Acids In The Given Samples By

Xanthoproteic Test.

PRINCIPLE: Aromatic amino acids, such as Phenyl alanine, tyrosine and

tryptophan, respond to this test. In the presence of concentrated nitric acid, the

aromatic phenyl ring gets nitrated to give yellow colored nitro-derivatives. At

alkaline pH the color changes to orange due to the ionization of the phenolic

group. Protein containing these amino acid also give a positive response to this

test.

MATERIALS AND REAGENTS:

1) Conc.HNO3

2) NaOH solution (40%, w/v): Dissolve 40gm of NaOH in water and make the final

volume to 100 ml.

3) Test solution: Prepare separate solution containing 0.5% of amino acid like

tyrosine, glycine, tryptophan, phenylalanine, lysine etc.




34

PROCEDURE: To 1ml of the amino acid solution taken in a test tube, add few

drops of nitric acid and vortex the contents. Boil the contents over a Bunsen flame

or in water bath, using a test tube holder, for few minutes. Cool the test tube

under running tap water and add few drops of alkali.Note whether the mixture

turns orange red in colour. Appearance of orange red colour denotes presence of

aromatic amino acid.

OBSERVATION TABLE:


1. 0.5% Glycine

2. 0.5% Trytophan

3. 0.5% Lysine

4. 0.5% Tyrosine





35

Experiment No.13

AIM: To Detect The Presence Of Amino Acids (Containing Hydroxybenzene

Radical) In The Given Samples By Millon’s Test.

PRINCIPLE: Phenolic amino acids such as Tyrosine and its derivatives respond to

this test. Compounds with a hydroxybenzene radical react with Millon’s reagent

to form a red colored complex. Millon’s reagent is a solution of mercuric sulphate

in sulphuric acid.

Hg + 4HNO3 Hg(NO3)2 + 2NO2+ 2H2O

REAGENTS:

1) Millon’s regent (15%W/V mercuric sulphate in 6N sulphuric acid)

2) Sodium nitrite (5%W/V) in distilled water ( to be freshly prepared)

3) 1mg/ml solution of glycine, casein & bovine serum albumin.

PROCEDURE: To 1ml of the amino acid solution in a test tube, add few drops of

millon’s reagent and vortex. Boil the contents over a Bunsen flame for 3 – 5

minutes. Cool the contents under running tap water and add few drops of sodium

nitrite solution. A positive reaction will also be obtained for the proteins which

contain tyrosine.




36

OBSERVATION TABLE:


1. 0.5% Glycine

2. 0.5% BSA

3. 0.5% Casein

4. 0.5% Tyrosine





37

Experiment No.14

AIM: To Detect The Presence Of Amino Acids (Containing Guanidium Group) In

The Given Samples By Sakaguchi’s Test.

PRINCIPLE: Under alkaline condition, α- naphthol (1-hydroxy naphthalene) reacts

with a mono – substituted guanidine compound like arginine, which upon

treatment with hypobromite or hypochlorite, produces a characteristic red color.

REAGENTS:

1) Amino acids: 0.5% solution of amino acids like glycine, arginine, lysine etc.

2) 0.5% urea solution

3) NaOH 40% (w/v)

4) α naphthol: 1% (w/v) in alcohol

5) Hypobromite solution (To be freshly prepared) : -Take 100 of 5%(W/V) sodium

hydroxide solution in a glass reagent bottle and add 1ml of pre chilled liquid

bromine, using a pro pipette. Shake the contents till bromine dissolves)

PROCEDURE: To 1 ml of prechilled amino acid solution and few drops of NaOH is

mixed and 2 drops of alpha naphthol is added. Mix thoroughly and add 4-5 drops

of hypobromite reagent and observe for the formation of red colour which would

indicate the presence of arginine or a guanidium compound.




38

OBSERVATION TABLE:


1. 0.5% Glycine

2. 0.5% Lysine

3. 0.5% Urea

4. 0.5% Arginine





39




40

Differences Encountered In A Real Laboratory:


necessarily applicable in a virtual lab.


lab, switch on the exhaust fan and make sure that all the reagents required for


the components shown in the reagent preparation.

2. Care should be taken while handling reagents like Conc. Sulphuric acid and

Hydrochloric acid. These concentrated acids should be opened and used only in

a FUMEHOOD. These concentrated acids cause severe burns and on inhaling

can cause damage to the lining of the lungs.

3. Reagents like Ninhydrin reagent, sulphanilic acid, isatin reagent, bromin,

Sodium nitroprusside should also be handled with care. Accidental spill of these

reagent will cause burns and itches. Wash the spilled area with cold water and

inform the lab assistant immediately.

4. Make sure that the waterbath is set to the proper temperature before starting

with the experiment.

5. Take care while heating the sample over the flame.

6. In Xanthoproteic test, results can be observed clearly on boiling the contents in

a waterbath.

7. The development of colors will depend upon the quality of the reagents

prepared.

8. Wipe the lab bench after the experiment is completed.

9. Make sure to switch off the waterbath and the exhaust fans before leaving the

lab.




41

EXPERIMENT NO.15

AIM: Test For Solubility Of Given Lipid Sample.

PRINCIPLE:

The test is based on the property of solubility of lipids in organic solvents and

insolubility in water. The oil will float on water because of lesser specific gravity.

REAGENTS:

1) Lipid sample

2) Different solvents – water, ethanol, acetone, chloroform & ether

PROCEDURE:

Place 5 drops of and oil or a small sample of your lipid into each of three separate

test tubes. To the first tube add 5 ml. of water, to the second 5 ml. of ethanol, to

the third 5 ml. of acetone, to the fourth 5 ml. of chloroform and to the firth add 5

ml. of ether. Shake each tube well and allow to stand for a few minutes. Observe

whether solution or emulsification has occurred.

OBSERVATION TABLE:

Si.No. Solvents Final Observation Interpretation

1. Water

2. Ethanol

3. Acetone

4. Chloroform

5. Ether





42

EXPERIMENT NO.16

AIM: Acrolein Test For The Presence Of Glycerol.

PRINCIPLE: When glycerol is heated with potassium bisulphate or concentrated

H2SO4, dehydration occurs and aldehyde Acrolein formed which has characteristic

odour. This test responds to glycerol free or linked as an ester.

CH2 – OH CH2

CH – OH CH + 2H2O

CH2 – OH CHO

Glycerol Acrolein

MATERIALS:

1. Test compounds ( Oil or fat ,Oleic acid)

2. Potassium bisulphate or conc. H2SO4

PROCEDURE:

1. Place 5 drops of test compound in a clean and dry test tube

2. Add 1 ml of conc. H2SO4 carefully. Or 1.0 g of KHSO4

3. Heat the test tube directly.

4. Note the characteristic pungent odour of Acrolein.


Heat

KHSO4 or Conc.H2SO4




43

EXPERIMENT NO.17

AIM: Zak Test For The Presence Of Cholesterol.

PRINCIPLE: This test is used for determination of cholesterol in blood.

MATERIALS:

1) 0.2 g cholesterol in 1ml of conc. acetic acid

2) Ferric chloride

3) Conc. Acetic acid

4) Conc. Sulfuric acid

PROCEDURE:

1. Place 0.5 ml of prepared cholesterol solution in a dry test tube.

2. Add 2 ml of colored solution ( mixture of 10% ferric chloride , Conc.

CH3COOH and Conc. H2SO4)

3. Observe appearance deep red which refers to existence of cholesterol.





44

Section: III

Quantitative Analysis Of Quantitative Analysis Of Quantitative Analysis Of Quantitative Analysis Of

BiomoleculesBiomoleculesBiomoleculesBiomolecules




45

EXPERIMENT NO.18

AIM: Estimation Of Carbohydrates (Total And Reducing Sugars, Sucrose And

Starch By Ferricyanide Method (Titrematric Method).

PRINCIPLE:

Alkaline potassium ferricyanide oxidizes sugars. This method is based on

reduction of the residual potassium ferricyanide by KI and the unreacted KI is

volumetrically measured by titration against Na2S2O3. The chemical reactions

involved are as follows:

2K2 [Fe (CN) 6] + 2KOH 2K4 [Fe (CN)6] + H2O + ½ O2

CH2OH (CHOH) 4 CHO + ½ O2 CH2OH (CHOH) 4 COOH

Glucose Gluconic acid

Excess of ferricyanide reacts with KI

KI [Fe (CN)6] + KI K4 [Fe(CN)6]2 + I

3ZnSO4 + 2K4 [Fe (CN)6] K2Zn3[Fe(CN)6]2 + 3K2SO4

Potassium zinc ferrocynide

3I2 + 6OH 5I- + IO3

- + 3H2O

5I- + IO3

- + 6H

+ 3I2 + 3H2O

2Na2S2O3 + I2 Na2 S4 O6 + 2NaI

Sodium tetrathionate


1. Burette

2. Boiling water bath.

3. Potassium ferricyanide: Dissolve 8.25g potassium ferricyanide and 10.6g

anhydrous sodium carbonate in 1 L of distilled water.




46

4. Iodine solution: Prepare by dissolving 12.6 g KI, 25 g ZnSO4 and 125 g NaCl in

500 ml distilled water. Filter and store in colored bottle.

5. Sodium thiosulphate solution: Make 0.01 N sodium thiosulphate solutions by

dissolving 2.5069 g of sodium thiosulphate in 1 L of distilled water.

6. Starch indicator solution: Suspend 1 g soluble starch in 20 ml of distilled water

and then add 60 ml of boiling water. Add 20g NaCl to this solution and make

the volume 100ml.

7. 5% glacial acetic acid.

PROCEDURE:

1. Take 5ml of potassium ferricyanide and 5 ml of aliquot of the sample extract in

a test tube, heat for 15 min in boiling water bath and then cool it.

2. Add 5 ml of iodine-solution followed by 3 ml of 5% glacial acetic acid. The

excess iodide is titrated against 0.01 N Na2S2O3 till the colour of the solution

turns pale yellow. Now add starch indicator solution, upon which the colour

will change to blue.

3. Complete the titration till disappearance of blue colour.

4. Run blank taking water instead of sugar solution or sample aliquot and proceed

in the same manner. Volume of Na2S2O3 used for the sample is deducted from

that consumed for the blank.

CALCULATIONS:

The amount of reducing sugars is calculated from the following relationship:

mg of reducing sugar in 5 ml of sample extract = µ (x + 0.05)

Where, µ = 0.338

x= vol. of 0.01 N Na2S2O3 used for sample, i.e.

Vol. of Na2S2O3 used in blank – Vol. used in sample.





47

Experiment No.19

AIM: To Estimate Protein Quantitatively In The Given Sample By Lowry’S

Method.

PRINCIPLE: The Lowry protein assay is named after Oliver H. Lowry, who

developed and introduced it (Lowry, et al., 1951). The phenolic group of tyrosine

and trytophan residues (amino acid) in a protein will produce a blue purple color

complex , with maximum absorption in the region of 660 nm wavelength, with

Folin- Ciocalteau reagent which consists of sodium tungstate molybdate and

phosphate. Thus the intensity of color depends on the amount of these aromatic

amino acids present and will thus vary for different proteins. Most proteins

estimation techniques use Bovin Serum Albumin (BSA) universally as a standard

protein, because of its low cost, high purity and ready availability.

The –CO-NH- (peptide bonds) in polypeptide chain reacts with copper sulphate in

an alkaline medium to give a blue coloured complex. In addition, tyrosine &

tryptophan residues of proteins cause reduction of the phosphomolybdate &

phosphotungstate components of the Folin-Ciocalteau reagent to give bluish

products which contribute towards enhancing the sensitivity of this method. It is,

however, important to remember that several compounds like EDTA, Tris,

carbohydrates, NH+

4, K+, Mg

++ ions, thiol reagents, phenols etc. interfere with the

colour development & it should be ensured that such substances are not present

in sample preparations. The incubation time is very critical for a reproducible

assay. The reaction is also dependent on pH and a working range of pH 9 to 10.5 is

essential.

REAGENTS:

1) Reagent A: 2% Na2CO3 solution was prepared in 0.1 N NaOH

2) Reagent B: 1% CuSO4.5H2O (prepared in water)

3) Reagent C: 2% sodium potassium tartarate (prepared in water)

4) Reagent D: 1.0 ml of reagent B and 1.0 ml of reagent C were mixed with 98.0 ml

of reagent A just prior to use.

5) Reagent E: 1N Folin-ciocalteau’s reagent prepared by diluting the commercially

available reagent (2 N) with equal volume of distilled water at the time of use




48

6) Reagent F: BSA standard protein solution (1 mg BSA/ mL of distilled water)

PROCEDURE: [Run triplicate determination for all samples.]

1) Different dilutions of BSA solutions are prepared by mixing stock BSA solution

(1 mg/ ml) and water in the test tube as given in the table. The final volume in

each of the test tubes is 1 ml. The BSA range is 0.01 to 0.10 mg/ ml.

2) Add 3.0 ml of freshly prepared reagent D (analytical reagent). Mix the solutions

well.

3) This solution is incubated at room temperature for 10 mins.

4) Then add 0.3 ml of reagent E to each tube and incubate for 30 min. Zero the

colorimeter with blank and take the optical density (measure the absorbance)

at 660 nm.

5) Plot the absorbance against protein concentration to get a standard calibration

curve.

6) Check the absorbance of unknown sample and determine the concentration of

the unknown sample using the standard curve plotted above.

BSA

(µL)

Water (µL) Sample conc.

(mg/mL)

Reagent D (mL) Reagent E (mL) O.D.

660 nm

0 1000 3 0.3

10 990 3 0.3

20 980 3 0.3

30 970 3 0.3

40 960 3 0.3

50 950 3 0.3

60 940 3 0.3

70 930 3 0.3

80 920 3 0.3

90 910 3 0.3

100 900 3 0.3




49

The protocol requires that the Folin phenol reagent be added to each tube

precisely at the end of the ten minute incubation. At the alkaline pH of the Lowry

reagent, the Folin phenol reagent is almost immediately inactivated. Therefore, it

is best to add the Folin phenol reagent at the precise time while simultaneously

mixing each tube. Because this is somewhat cumbersome, some practice is

required to obtain consistent results. This also limits the total number of samples

that can be assayed in a single run.





50

Experiment No.20

AIM: To Determine The Acid Value Of The Given Fats Or Oil Sample.

PRINCIPLE: Different fat sample may contain varying amount of fatty acids. In

addition, the fats often become rancid during storage and this rancidity is

chemical or enzymatic hydrolysis of fats into free acids and glycerol the amount of

free fatty acids can be determined volumetrically by treating the sample with

potassium hydroxide. The acidity of fats and oils is expressed as its acid value or

number which is defined as mg KOH required to neutralize the free fatty acid

present in 1gm of fat or oil. The amount of free acids present or acid value of fat is

a useful parameter which gives an indication about the age and extent of its

deterioration.


1) Burette

2) Conical flask.

3) Test compounds (olive oil, butter, margarine etc; fresh and samples that have

been stored at room temperature for several days may be used for

comparison)

4) 1% phenolphthalein solution in 95% alcohol.

5) 0.1N potassium hydroxide: Weigh 5.6g of KOH and dissolve it in distilled water

and make the final volume to 1L. Standardize this solution by titrating it with a

known volume of 0.1N oxalic acid (prepare by taking 630mg oxalic acid in

100ml water) using phenolphthalein as indicator till a permanent pink colour

appears. Calculate the actual normality (N2) of KOH solution from equation

N1V1 = N2V2, where N1 and v1 are normality and volume of oxalic acid taken for

titration and V2 is the volume of KOH solution used.

6) Fat solvent (95% ethanol : ether 1:1, v/v)

PROCEDURE:

1) Take 5g of fat sample in a conical flask and add 25ml of fat solvents (reagent

no.6) to it .Shake well and a few drops of phenolphthalein solution and again

mix the content thoroughly.




51

2) Titrate the above solution with 0.1N KOH until a faint pink colour persists for

20-30sec.

3) Note the volume of KOH used.

4) Repeat the steps 1-3 with a blank (reagent no.6) which does not contain any fat

sample.

CALCUTATION:

0.1N KOH solution used for blank = xml

0.1N KOH solution used for sample = yml

Titer value for sample = (y-x) ml

Acid value (mg KOH/g fat) =

1ml of 1N KOH contains 56.1mg of KOH. Hence a factor of 56.1 is incorporated in

the numerator in the above equation to obtain weight of KOH from the volume of

0.1N KOH solution used during this titration.





52

Experiment No. 21

AIM: To Determine Of Saponification Value Of The Given Fats Or Oil Sample.

THEORY:

Saponification is

the hydrolysis of

fats or oils

under basic

conditions to

afford glycerol

and the salt of

the

corresponding fatty acid. Saponification literally means "soap making". It is

important to the industrial user to know the amount of free fatty acid present,

since this determines in large measure the refining loss. The amount of free fatty

acid is estimated by determining the quantity of alkali that must be added to the

fat to render it neutral. This is done by warming a known amount of the fat with

strong aqueous caustic soda solution, which converts the free fatty acid into soap.

This soap is then removed and the amount of fat remaining is then determined.

The loss is estimated by subtracting this amount from the amount of fat originally

taken for the test.

The saponification number is the number of milligrams of potassium hydroxide

required to neutralize the fatty acids resulting from the complete hydrolysis of 1g

of fat. It gives information concerning the character of the fatty acids of the fat-

the longer the carbon chain, the less acid is liberated per gram of fat hydrolysed.

It is also considered as a measure of the average molecular weight (or chain

length) of all the fatty acids present. The long chain fatty acids found in fats have

low saponification value because they have a relatively fewer number of

carboxylic functional groups per unit mass of the fat and therefore high molecular

weight.

PRINCIPLE: Fats (triglycerides) upon alkaline hydrolysis (either with KOH or NaOH)

yield glycerol and potassium or sodium salts of fatty acids (soap).




53

The procedure involves reflexing of known amount of fat or oils with a fixed an

excess of alcoholic KOH. The amount of KOH remaining after hydrolysis is

determined by back titrating with standardized 0.5N HCl and amount of KOH

utilized for saponification can thus be calculated.

MATERIALS REQUIRED:

1) Fats and Oils [coconut oil, sunflower oil]

2) Conical Flask

3) 100ml beaker

4) Weigh Balance

5) Dropper

6) Reflux condenser

7) Boiling Water bath

8) Glass pipette (25ml)

9) Burette

REAGENTS REQUIRED:

1) Ethanolic KOH(95% ethanol, v/v)

2) Potassium hydroxide [0.5N]

3) Fat solvent

4) Hydrochloric acid[0.5N]




54

5) Phenolphthalein indicator

PROCEDURE:

1) Weigh 1g of fat in a tared beaker and dissolve in about 3ml of the fat solvent

[ethanol /ether mixture].

2) Quantitatively transfer the contents of the beaker three times with a further

7ml of the solvent.

3) Add 25ml of 0.5N alcoholic KOH and mix well, attach this to a reflux

condenser.

4) Set up another reflux condenser as the blank with all other reagents present

except the fat.

5) Place both the flask on a boiling water bath for 30 minutes.

6) Cool the flasks to room temperature.

7) Now add phenolphthalein indicator to both the flasks and titrate with 0.5N

HCl.

8) Note down the endpoint of blank and test.

9) The difference between the blank and test reading gives the number of

millilitres of 0.5N KOH required to saponify 1g of fat.

10) Calculate the saponification value using the formula:

Saponification value or number of fat = mg of KOH consumed by 1g of fat.

Weight of KOH = Normality of KOH x Equivalent weight x volume of KOH in litres

Volume of KOH consumed by 1g fat = [Blank – test]ml

CALCULATIONS:

Volume of 0.5N KOH used for titrating blank= x ml

Volume of 0.5N KOH used for titrating test sample= y ml

Titre value of sample = (x-y) ml

Saponification value = 28.05x titre value

Wt. of sample (g)




55





56

Differences Encountered In a Real Laboratory:







2. Care should be taken while handling reagents like potassium hydroxide and

hydrochloric acid. Accidental spill of these reagents will cause severe itching

sensation. Wash the spilled area with cold water and inform the lab assistant

immediately.

3. Caution should be taken while attaching the reflux condensors to the conical

flask.

4. Make sure that the waterbath is set to 100 degree celsius and the reflux

condensors are set up with proper settings before starting with the

experiment.

5. The endpoint point of titration should be carefully observed as the

disappearance of pink colour to white color.

6. After the experiment, switch off the waterbath and carefully remove the reflux

condensors.

7. After completing the experiment, clean the glass wares and wipe the lab bench.

8. Switch off the exhaust fans.




57

Section: IV

Chromatographic TechniquesChromatographic TechniquesChromatographic TechniquesChromatographic Techniques




58

EXPERIMENT NO.22

AIM: Separation And Identification Of Amino Acids By Ascending Paper

Chromatography.

THEORY:

Chromatography is a common technique for separating chemical substances. The

prefix “chroma,” which suggests “color,” comes from the fact that some of the

earliest applications of chromatography were to separate components of the

green pigment, chlorophyll. In this experiment you will use chromatography to

separate and identify amino acids, the building blocks of proteins.

Chromatography is a common technique for separating chemical substances. The

prefix “chroma,” which suggests “color,” comes from the fact that some of the

earliest applications of chromatography were to separate components of the

green pigment, chlorophyll. You may have already used this method to separate

the colored components in ink. In this experiment you will use chromatography to

separate and identify amino acids, the building blocks of proteins.

The term “paper chromatography” used in this experiment’s title identifies the

composition of the stationary phase. The compositions of the stationary and

mobile phases define a specific chromatographic method. Indeed, many different

combinations are possible. However, all of the methods are based on the rate at

which the analyzed substances migrate while in simultaneous contact with the

stationary and mobile phases. The relative affinity of a substance for each phase

depends on properties such as molecular weight, structure and shape of the

molecule, and the polarity of the molecule.

PRINCIPLE:

In this experiment, very small volumes of solutions containing amino acids will be

applied (this process is sometimes called “spotting”) at the bottom of a

rectangular piece of filter paper. For ready comparison of each trial, it is vital that

each solution be applied on the same starting line. After the solutions have been

applied, the paper will be rolled into a cylinder and placed in a beaker that

contains a few milliliters of the liquid mobile phase. For this separation, a solution

containing n-butanol, water and acetic acid is the optimum mobile phase. As soon

as the paper is placed in the mobile phase, the solution (sometimes called the




59

eluting solvent) will begin to rise up the paper. This phenomenon is called

capillary action.

As the mobile phase rises on the paper it will eventually encounter the “spots” of

amino acids. The fate of each amino acid in the mixture now depends on the

affinity of each substance for the mobile and stationary phases. If an amino acid

has a higher affinity for the mobile phase than the stationary phase, it will tend to

travel with the solvent front and be relatively unimpeded by the filter paper. In

contrast, if the amino acid has a higher affinity for the paper than the solvent, it

will tend to “stick” to the paper and travel more slowly than the solvent front. It is

these differences in the amino acid affinities that lead to their separation on the

paper. The affinities of these amino acids for the mobile phase can be correlated

to the solubility of the different amino acids in the solvent (i.e., an amino acid that

is highly soluble in the eluting solvent will have a higher affinity for the mobile

phase than an amino acid that is less soluble in the solvent.).

When the solvent front comes near the top of the filter paper, the paper is

removed from the beaker and allowed to dry. At this point, the various amino

acids are invisible. The acids can be visualized by spraying the paper with a

compound called ninhydrin. Ninhydrin reacts with amino acids to form a blue-

violet compound. Therefore, the sprayed filter paper should show a number of

spots, each one corresponding to an amino acid. The further the spot from the

starting line, the higher the affinity of the amino acid for the mobile phase and

the faster its migration.

The relative extent to which solute molecules move in a chromatography

experiment is indicated by Rf values. The R

f value for a component is defined as

the ratio of the distance moved by that particular component divided by the

distance moved by the solvent. Figure 1 represents the migration of two

components. Measurements are made from the line on which the original

samples were applied to the center of the migrated spot. In the figure, dA

is the

distance traveled by component A, dB

is the distance traveled by component B,

and dsolv

is the distance traveled by the eluting solution. In all three cases, the

travel time is the same.

Thus the Rf values for components A and B are

Rf(A) = d

A/d

solv R

f(B) = d

B/d

solv

Figure 1: Paper chromatography - migration of two components.




60

Note that Rf values can range from 0 to 1. In this example, R

f(A) is obviously larger

than Rf(B). Although R

f values are not exactly reproducible, they are reasonably

good guides for identifying the various amino acids. Paper chromatography is

most effective for the identification of unknown substances when known samples

are run on the same paper chromatograph with unknowns.

The separated amino acids are detected by spraying the air dried chromatogram

with ninhydrin reagent. All amino acids give purple or bluish purple colour on

reaction with ninhydrin except proline and hydroxylproline which give a yellow

coloured. The reactions leading to the formation of purple complexes are given

below:

Ninhydrin + Amino acid Hydrindantin + RCHO + NH3 + CO2

Ninhydrin + Ammonia + Hydrindantin Purple coloured product + 3H2O

MATERIALS AND REAGENTS

1) Whatman No. 1 filter paper sheet.

2) Micropipette / micro syringe.

3) Hair drier.

4) Sprayer.

5) Oven set at 105oC.

6) Chromatographic chamber saturated with solvent vapours.




61

7) Developing solvent: Take butanol, acetic acid and water in the ratio of 4:1:5 in a

separating funnel and mix it thoroughly. Allow the phases completely. Use the

lower aqueous phase for saturating the chamber.

PROCEDURE:

1. Obtain a sheet of filter paper, and draw a faint pencil line about 1 to 2 cm

from one of the long edges and parallel to that edge. This will be the bottom

of the chromatogram.

2. Mark off seven equally spaced points along this line. (They should be

separated by about 2 cm). Your samples will be applied to these spots. The

laboratory contains solutions of four identified amino acids and a sample of a

hydrolyzed protein. In addition, you will be given a numbered unknown that

will contain one or more of the known amino acids.

3. Dip the open end of a clean capillary into the solution to draw up a small

volume of the solution into the tube. Lightly and briefly touch the tube to the

paper and allow the sample to transfer. The spot should be about 2-3 mm in

diameter. Place one spot of each of the four known amino acids on the

separate points that you previously marked on the filter paper. In addition,

apply samples of your unknown to two of the points. Be careful not to

contaminate either the solutions or the spots.

4. Label each spot (with pencil and below the starting line) to indicate its

identity. Finally, it’s a good idea to avoid getting fingerprints on the

chromatographic paper.

5. When you have finished spotting your paper, allow it to dry by waving it in the

air or using a heat lamp or hair dryer. (Don’t get it too hot.)

6. Meanwhile, in the hood, pour about 15-20 mL of the eluting solution (n-

butanol and acetic acid) into a clean, dry 600 mL beaker and cover the beaker

with a watch glass or plastic wrap.

7. When the sample spots have dried, roll the paper into a cylinder, with the

short sides almost touching. Use a bit of “Scotch” tape along the top of the

paper to hold the cylinder together.

8. Evenly lower the paper cylinder, sample side down, into the beaker. The

solvent will wet the paper, but the sample spots should not be immersed. In

addition, the paper should not touch the walls of the beaker.




62

9. At this point, cover the beaker with a watch glass or plastic wrap and place the

beaker in the hood.

10. When the solvent front gets within about 1 or 2 cm of the top of the paper (in

perhaps about 2 hrs), remove the paper, use a pencil to mark the solvent front

at several points, unroll the cylinder, and let the chromatography paper dry in

the hood.

11. When the paper is dry, spray it with ninhydrin reagent.

12. Allow the paper to dry, perhaps using the hair dryer, heat lamp, or an oven at

about 100o

C, but don’t overcook it!

13. When the chromatographic paper has fully dried, outline the spots, mark the

centers of each of the spots, and note their colors. (Not all amino acids give

the same color with ninhydrin).

14. Measure and record the distances the solvent and each of the amino acids

traveled from the origin.

15. Use these distances to calculate Rf values for each sample.

Comparison of the spots should enable you to identify the amino acid(s) present

in your unknown sample.

CALCULATION:

Rf value can be calculated using the formula:





63

EXPERIMENT NO.23

AIM: Separation And Identification Of Amino Acids By Thin Layer

Chromatography.

THEORY:

Thin layer chromatographic (TLC) technique readily provides qualitative

information and with careful attention to details, it is possible to obtain

quantitative data. Thin layer chromatography is a technique used to separate and

identify compounds of interest. A TLC plate is made up of a thin layer of silica

adhered to glass or aluminum for support. The silica gel acts as the stationary

phase and the solvent mixture acts as the mobile phase. In the ideal solvent

system the compounds of interest are soluble to different degrees. Separation

results from the partition equilibrium of the components in the mixture.

In the simplest form of the technique, a narrow zone or spot of the sample

mixture to be separated is applied near one end of the TLC plate and allowed to

dry. The strip or plate is then placed with this end dipping in to the solvent

mixture, taking care that the sample spot/zone is not immersed in the solvent. As

the solvent moves towards the other end of the strip, the test mixture separates

into various components. This is called as the development of TLC plates. The

separation depends on several factors; (a)solubility: the more soluble a

compound is in a solvent, the faster it will move up the plate. (b) attractions

between the compound and the silica, the more the compound interacts with

silica, the lesser it moves, (c) size of the compound, the larger the compound the

slower it moves up the plate.

The plate is removed after an optimal development time and dried and the

spots/zones are detected using a suitable location reagent. An important

characteristic used in thin layer chromatography is Rf value.

The plate is removed after an optimal development time and dried and the

spots/zones are detected using a suitable location reagent. An important

characteristic used in thin layer chromatography is Rf value.




64

Chromatographic Separation of Amino acids:

The present experiment employs the technique of thin layer chromatography to

separate the amino acids in a given mixture.

All 20 of the common amino acids [standard amino acids] are a-amino acids. They

have a carboxyl group and an amino group bonded to the same carbon atom (α-

carbon). They differ from each other in their side chains, or R groups, which vary

in structure, size, and electric charge. The interaction of the amino acids with the

stationary phase like silica varies depending on their 'R' groups. The amino acid

that interacts strongly with silica will be carried by the solvent to a small distance,

whereas the one with less interaction will be moved further. By running controls

[known compounds] alongside, it is possible to identify the components of the

mixture.

Since amino acids are colourless compounds, ninhydrin is used for detecting

them. To identify this, after development, the TLC plate is sprayed with ninhydrin

reagent and dried in an oven, at 105°C for about 5 minutes. Ninhydrin reacts with

α- amino acids that results in purple coloured spots [due to the formation of the

complex - Rheuman's purple]. Rf values can be calculated and compared with the

reference values to identify the amino acids. [The Rf value for each known

compound should remain the same provided the development of plate is done

with the same solvent, type of TLC plates, method of spotting and in exactly the

same conditions].

MATERIALS REQUIRED:

REAGENTS:




65

1. 2% solution of individual amino acids.

2. Solvent mixture of normal butanol, acetic acid and water in the ratio 12:3:5 by

volume.

3. Ninhydrin reagent.

Requirements:

1. TLC plate.

2. TLC chamber.

3. Capillary tubes.

4. Reagent spray bottle.

5. Conical flasks.

6. Beakers.

Procedure:

1. Pour the solvent mixture in to the TLC chamber and close the chamber.

2. The chamber should not be disturbed for about 30 minutes so that the

atmosphere in the jar becomes saturated with the solvent.

3. Cut the plate to the correct size and using a pencil (never ever use a pen)

gently draw a straight line across the plate approximately 2 cm from the

bottom.

4. Using a capillary tube, a minute drop of amino acid is spotted on the line.

5. Allow the spot to dry.

6. Spot the second amino acid on the plate [enough space should be provided

between the spots].

7. Repeat the above step for spotting the unknown acid.

8. Place the plate in the TLC chamber as evenly as possible and lean it against

the side(immerse the plate such that the line is above the solvent). Allow

capillary action to draw the solvent up the plate until it is approximately 1 cm

from the end.




66

9. Remove the plate and immediately draw a pencil line across the solvent top.

10. Under a hood dry the plate with the aid of a blow dryer.

11. Spray the dry plate with ninhydrin reagent.

12. Dry the plates in hot air oven at 105°C for 5 min. [Ninhydrin will react with the

faded spots of amino acids and make them visible as purple coloured spots.]

13. After some time, mark the center of the spots, then measure the distance of

the center of the spots from the origin and calculate the Rf values.

Rf value can be calculated using the formula:

The Rf values with butanol-acetic acid- water solvent are as follows: alanine 0.24,

glutamic acid 0.25, glycine 0.2, leucine 0.58, valine 0.4, lysine 0.58, tyrosine 0.42.





67

Differences Encountered In A Real Laboratory:







2. Care should be taken while handling reagents like Ninhydrin reagent. This

reagent is a strong oxidizing agent and should not be inhaled or spilled on

hands or other body parts. Accidental spill of this reagent will cause severe

itching sensation. Wash the spilled area with cold water and inform the lab

assistant immediately.

3. Hold the TLC plates by their side. Ensure that you do not touch the developing

part of the TLC plate, because your finger prints will also get developed causing

the result to be unclear.

4. Make certain that the spots applied to the plate are above the surface of the

eluting solvent.

5. Before applying the second spot make sure that the previously applied spot is

dried.

6. Spot the components with proper space in between.

7. Ensure that the chamber is saturated with the solvent vapour before you place

the TLC plate in it.

8. Give enough time for the solvent to advance up the plate.

9. The top of the solvent must not advance up to or beyond the edge of the

plates.




68

Section: V

Electrophoretic TechniquesElectrophoretic TechniquesElectrophoretic TechniquesElectrophoretic Techniques




69

Experiment No.24

AIM: To Conduct Agarose Gel Electrophoresis.

PRINCIPLE: DNA molecules are negatively charged at neutral or alkaline pH and

migrate towards anode when an electric field is applied. Charge/mass ratio in

nucleic acid is unity, thus migration largely occurs on the basis of molecular size of

DNA molecules.


1. Mini gel Horizontal Agarose Gel electrophoresis unit. Comprises of:-

i) Gel casting plate

ii) Electrophoretic tank

iii) Comb

iv) Electrophoretic leads

2. Adhesive tape

3. Power pack

4. U.V Transilluminator with camera

5. Micropipette

6. Gloves

7. Tris acetate buffer (TAE) stock solution (5X): A five fold concentrated TAE buffer

stock solution. Consist of:

i) Tris base : 24.2g

ii) Glacial Acetic acid : 5.71ml

iii) 0.5M Acetic acid : 10.0ml

Adjust pH to 8.0 and add water to make 1lilre. Dilute 5 times to obtain working

washing buffer (1X).

8. 0.8% Agarose in 1X TAE Buffer: Dissolve 0.4g agarose in 50ml of 1X TAE buffer

by boiling and maintaining it at 50oc to be used.




70

9. Gel loading solution: 1% glycerol and 0.025% bromophenol blue in water.

10. Ethidium bromide: Dissolve 10mg ethidium bromide per ml. of 1x TAE buffer.

11. Isolated DNA sample

PROCEDURE:

1. Take a clean dry gel casting plate and make gel mould using an adhesive tape

along the sides of the plate to prevent running off the material to be poured

on the plate.

2. Pour 1% agarose solution kept at 50oC onto casting plate, immediately place

the comb about 1cm from one end of the plate ensuring that teeth of the

comb do not touch the glass plate.

3. Allow a firm layer of gel formation. Remove the comb and tape surrounding

the plate carefully and transfer the gel plate to the electrophoretic tank such

that wells are towards the cathode.

4. Pour 1X TAE buffer into the tank until the gel is completely submerged;

connect the electrodes to the power supply.

5. Load the isolated DNA preparation into well with the help of the micropipette.

6. Turn on the power supply and run at 100v. Monitor the progress of

bromophenol blue (tracking dye) during electrophoresis.

7. Turn OFF the power supply when tracking dye has reached the opposite side

of the gel.

8. Transfer the gel from the casting plate onto a UV- Transparent thick plastic

sheet and place it in staining tray containing ethidium bromide solution stain

for 20-30min.

9. For destaining the gel, place it in water for 15-20min.

10. Now place the gel along with UV transparent sheet on a UV transilluminator

and view the gel in UV light for presence of orange coloured bands.

11. Gel should be photographed to keep the permanent store.

RESULT & INTERPRETATION:




71

EXPERIMENT NO.25

AIM: To Perform Poly Acrylamide Gel Electrophoresis (PAGE).

PRINCIPLE: Electrophoresis is the migration of charged molecule in solution

through a support matrix in response to an electric field. Rate of migration

depends on the strength of the field; on the net charge, size and shape of the

molecules and also on the ionic strength, viscosity and temperature of the matrix.

As an analytical tool, electrophoresis is simple, rapid and highly sensitive.

Polyacrylamide gels are prepared by copolymerization of acrylamide monomer

(CH2=CH-CO-NH2) with a cross linking agent in the presence of the catalyst

accelerator and chain initiator mixture. The relative proportion of acrylamide

monomer to cross-linking agents determine the porosity of the gel. Separation of

protein is carried out using gels ranging from 5-20% of acrylamide. Discontinuity

of the buffer pH and gel concentration is employed to effect band sharpening at

the end of electrophoresis. Polyacrylamide is the medium of choice where high

resolution electrophoresis on the basis of charge and molecular size is required.

Other advantage includes its minimal adsorption capacity, lack of electro osmosis,

preparation of zymogram etc. PAGE is also used in specialized electrophoretic

system such as SDS-PAGE and isoelectric focusing. Sodium dodecyle sulphate

(SDS) is used to induce uniform negative charges on the protein molecule which

itself is charged. In SDS-PAGE the protein molecules are preferentially separated

on the basis of their molecular weight where as in native PAGE separation of

molecule depends on the charge as well as the mass of the entity.

The extent of purification and number of protein components are monitored by

SDS-PAGE. It is performed using 5% stacking gel and 12% resolving gel having pH

of 6.8to 8.8 respectively following methods of Laemmli (1994). Sample is

prepared by mixing protein solution and denaturing sample buffer at 1:1 ratio and

Tris-glycine buffer of pH 8.3 is used as electrode buffer. Separation is performed

at 15mA fixed current until the tracking dye reaches near the end of gel.

Gel is stained by silver staining following the methods of Blum et al, (1987).

Proteins are fixed in gel by overnight treatment with a solution containing 50%

methanol, 12% acetic acid, 0.15% HCHO. Gel is washed with double distilled water

and is incubated in freshly prepared sensitizing solution (0.125% glutaraldehyde,

0.2% sodium thiosulphate, 7% sodium acetate). After 40 minutes the sensitizing

solution is drained off and the gel is rinsed with double distilled water. Gel is

treated with 0.25% silver nitrate solution for 30 minutes, washed and is




72

incubated in freshly prepared developing solution (3% sodium carbonate, 0.015%

HCHO) till the bands appear. Immediately after appearance of bands developing

solution is removed and the gel is immersed in stop solution containing 1.5%

EDTA. Longer incubation with developing solution may lead to background

staining.

1. NATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS (NATIVE-PAGE): Native-

PAGE is done using anionic system of Davis (1964).

REAGENTS USED:

• Acrylamide-bis-acrylamide solution (29.2:0.8): Dissolved 29.2 g acrylamide and

0.8 g N,N’-methylene-bis-acrylamide in distilled water and made up the volume

to 100 ml. Filtered this solution through Whatman filter paper and stored in a

brown bottle at 4oC.

• Resolving gel buffer stock (1.5 M Tris-HCl, pH 8.8): Dissolved 18.17 g of Tris

base in 60 ml distilled water and adjusted the pH of the solution to 8.8 with 1 N

HCl and the final volume was made to100 ml with distilled water. It is stored at

room temperature.

• Stacking gel buffer stock (1.0 M Tris buffer, pH 6.8): Dissolved 12.11 g of Tris

base in 60 ml distilled water and adjusted the pH to 6.8 and its volume is made

up to 100 ml with distilled water. It is stored at room temperature.

• Ammonium persulphate solution (1.5% w/v): The solution is prepared fresh by

dissolving 15.0 mg in 1.0 ml water.

• Reservoir buffer: 3.0 g Tris base and 14.4 g glycine are dissolved in 1000 ml

distilled water. The pH of the solution is adjusted to 8.3.

• Staining solution: Dissolved 250 mg Coomassie brilliant blue R-250 dye in a

solution containing 125 ml methanol and 25 ml glacial acetic acid. It’s volume is

adjusted to 250 ml with distilled water. It is filtered to remove any undissolved

material and stored at room temperature.

• Destaining solution: Mixed 50 ml methanol with 40 ml glacial acetic acid and

made up its volume to 100 ml with distilled water.

• TEMED: As supplied by the manufacturer




73

• Sample preparation: Sample was prepared by mixing the purified protein with

the sample buffer (1.0 M Tris-HCl, pH 6.8 containing 5% glycerol and 0.02%

bromophenol blue).

PROCEDURE: Properly cleaned and dried glass plates were tightly held with the

spacer bars on both sides. Resolving (12%) and stacking gel (2.5%) solutions for

polymerization were prepared as given in Table 3.1.

Table: Composition of different recipe of gels for Native-PAGE

Native Gel Recipe (for 3.0 % to 15% PAGE)

Gel % 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.5 10.0 12.5 15

Water ml 6.4 6.25 6.0 5.9 5.75 5.6 5.4 5.25 5.1 4.9 4.5 4.1 3.3 2.4

Lower Tris ml 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5

Acrylamide

(30%) + bis

Acrylamide

(8%)

ml 1.0 1.17 1.34 1.5 1.67 1.8

1

2.0

2 2.17 2.34 2.5 2.92 3.3 4.1 4.92

APS (10%) µL 85 87 87 80 75 75 70 70 62 45 45 45 45 45

TEMED µL 6.2 6.2 6.2 6.2 5.0 5.0 5.0 3.7 3.7 3.7 2.5 2.5 2.5 2.5

The solution of resolving gel is poured into a vertical slab and a few drops of

distilled water are layered on top of the gel solution to ensure the production of a

flat gel surface. The gel is allowed to polymerize for half an hour.

After polymerization of resolving gel, the water layer is removed and soaked off

with filter paper. The stacking gel solution is then poured and immediately the

comb is fixed at the top to make the wells for sample application. The stacking gel

is allowed to polymerize for half an hour. The comb is removed and the wells are

cleared thoroughly with reservoir buffer using a syringe so that no unpolymerized

acrylamide is left in the wells. The spacer fixed on the lower side is removed and

the lower and upper chambers of the apparatus are filled with reservoir buffer in

such a manner that no air bubble is formed between gel and buffer system. After

this, pre-electrophoresis is carried out at 10 mA for 15 min.

Protein samples are dissolved in sample buffer 1.0 M Tris-HCl (pH 6.8) containing

5% glycerol and 0.02% bromophenol blue and loaded into the wells using a




74

Hamilton syringe. The electrodes are connected to electrophoretic power supply

unit and run at 10 mA till the dye reached the end of stacking gel.

Coomassie Staining: After the electrophoresis, the gel is taken out and stained

using coomassie brilliant blue R-250 staining solution with constant shaking for 8

h to visualize protein bands. After staining, the gel is transferred to destaining

solution. The gel is destained with gentle shaking on a gel rocker by changing the

destaining solution several times till the gel background is clear. After destaining,

the gels are photographed and preserved in destaining solution with 10% glycerol

in dark and cool place.

Silver Staining: The protein bands in the gels are also stained by silver staining as

described by Blum et al, (1987). The gel is removed from the chamber and

transferred to the fixative solution (50% methanol and 7% acetic acid in water) for

2 h. The gel is soaked in Hypo solution (20 mg of sodium thiosulfate in 100 ml of

distilled water) for 1 min and then rinsed with distilled water three times for 1

min each. It is then transferred to staining solution (100 mg of AgNO3 in 100 ml of

distilled water and 75 µL of formaldehyde) and stained for 20 min. After proper

washing with distilled water, the gel is developed in a solution of 100 ml of

distilled water containing 2.0 g of Na2CO3 and 50 µL of formaldehyde. After the

development, the reaction is stopped by 0.1% citric acid.

2. SDS-PAGE (sodium dodecyl sulfate- poly acrylamide gel electrophoresis)

SDS-PAGE is carried out by the method of Laemmli (1970) with slight

modifications. acrylamide-bis-acrylamide solution, resolving gel buffer, stacking

gel buffer, ammonium persulphate, staining and destaining solutions, and

bromophenol blue are the same as used for Native- PAGE. The following

additional solutions were prepared for SDS-PAGE:

SDS (10%, w/v): Dissolved 1 g SDS in 10 ml of distilled water.

Reservoir buffer: 3.0 g Tris base, 14.4 g glycine and 1 g SDS are dissolved in

distilled water and its pH was adjusted to pH 8.3. The volume was made to 1 L

with distilled water.

Sample buffer (2x): It is prepared by mixing 2.5 ml of 1M Tris-HCl buffer (pH 6.8),

2.0 ml glycerol (20%), 0.4 g SDS, 1.0 ml β-mercaptoethanol and 0.4 ml of 1%

bromophenol blue and volume is made to 10.0 ml with distilled water.

Sample preparation: Purified enzyme solution is mixed with equal volume of

sample buffer (2x), boiled for 5 min and cooled prior to loading.




75

Molecular weight markers: A pre-stained mixture of SDS-PAGE molecular weight

markers viz. BSA (66 kDa), ovalbumin (46 kDa), pepsin (34.7 kDa), trypsinogen (24

kDa) and lysozyme (14.3 kDa) is used as supplied.

PROCEDURE: SDS-PAGE was performed using 12% resolving and 2.5% stacking

gel, the compositions of which are given in Table 3.2. The gels are prepared as

described for native PAGE. The sample containing 100-150µg protein was loaded

in to the wells. The standard SDS-PAGE molecular weight markers are co-

electrophoresed. The electrophoresis is carried out and gels are processed for

visualization of protein bands as described for native-PAGE. Molecular mass of

the purified enzyme protein is calculated using Gel Documentation system.

Table: Composition of different Recipe of gels for SDS-PAGE

Native Gel Recipe (for 3.0 % to 15% PAGE)

Gel % 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.5 10.0 12.5 15

Water ml 6.4 6.25 6.0 5.9 5.75 5.6 5.4 5.25 5.1 4.9 4.5 4.1 3.3 2.4

Lower Tris ml 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5

Acrylamide

(30%) + bis

Acrylamide

(8%)

ml 1.0 1.17 1.34 1.5 1.67 1.81 2.02 2.17 2.34 2.5 2.92 3.3 4.1 4.92

APS (10%) µL 85 87 87 80 75 75 70 70 62 45 45 45 45 45

TEMED µL 6.2 6.2 6.2 6.2 5.0 5.0 5.0 3.7 3.7 3.7 2.5 2.5 2.5 2.5




76

Section: VI

SpectroscopiSpectroscopiSpectroscopiSpectroscopic Techniquesc Techniquesc Techniquesc Techniques




77

EXPERIMENT NO.26

AIM: (a) Verify Beer’s Law And Apply It To Find The Concentration Of The Given

Unknown Solution.

(b) To Determine ʎmax (Wave Length Of Maximum Absorption) Of Solution Of

KMNO4 Using A Spectrophotometer.

THEORY:

When an electromagnetic radiation is passed through a sample, certain

characteristic wavelengths are absorbed by the sample. As a result the intensity

of the transmitted light is decreased. The measurement of the decrease in

intensity of radiation is the basis of spectrophotometer. Thus the

spectrophotometer compares the intensity of the transmitted light with that of

incident light.

The absorption of light by a substance is governed by certain laws.

According to the Beer Lambert’s law the intensity of the incident light is

proportional to the length of thickness of the absorbing medium and the

concentration of the solution,

Log Io/I = A = ε . c

Io = Intensity of incident light

I = Intensity of transmitted light

A = Absorbance

L = Thickness of the medium

c = Concentration in mol L-1

ε = Molar absorption coefficient

The molar absorption coefficient is the absorbance of a solution having unit

concentration (C = 1M) placed in a cell of unit thickness (l= 1cm). Absorbance is

also called Optical Density (OD)

The absorbance (OD) of a solution in a container of fixed path length is directly

proportional to the concentration of a solution. i .e

A = ε.c




78

A plot between absorbance and concentration is expected to be linear. Such a

straight line plot, passing through the origin, shows that Beer – Lambert’s law is

obeyed. This plot, known as calibration curve can be employed in finding the

concentration of a given solution.

REAGENTS: Distilled water, standard solution of KMnO4, tissue paper.

APPARATUS: UV – visible spectrophotometer, beaker.

SPECTROPHOTOMETER:

A spectrometer is a device which detects the percentage transmittance of light

radiation when light of certain intensity and frequency range is passed through

the sample. Thus the instrument compares the intensity of the transmitted light

with that of the incident light.

There are many spectrophotometers available for the visible range extending

from 3800- 7800 Ao.

Setting of the Spectrophotometer

1) Spectrophotometer should initially read zero on transmittance scale (T). if it

does not read zero, set it mechanically with adjusting knob.

2) Connect the instrument to the mains and put on the power switch.

3) Adjust the wavelength knob to the required wavelength region on scale.

4) Choose the position of wavelength switch correspondingly either to 340 –

400 nm or 400-960nm.

5) Adjust the meter needle on zero transmittance scale and 100 on O.D scale.

Working of the Spectrophotometer

6) Open the lid of the cell compartment and insert a cuvette containing the

blank solvent (distilled water). Close the lid.

7) Adjust the needle to 100% transmittance or zero optical density.

8) Remove the cuvette and close the lid tightly again. Empty the cuvette and

rinse it with the standard solution of KMnO4 (0.01IM). Fill it with standard

solution.




79

9) Now place the cuvette containing the standard solution in the cell

compartment. Note the O.D and transmittance.

10) Now change the wavelength by 20nm and note absorbance (OD) and

transmittance for each wavelength.

11) Plot a graph between wavelength measurement on the x-axis and

absorbance (OD) on the y-axis.

Verification of Beer’s law

12) Fix the wavelength at ʎmax position.

13) Prepare KMnO4 solution with concentration 0.2%, 0.5%, 1.0%, 1.5%, 2.0%,

2.5%, and 3.0% etc. (20ml each)

14) Note down the absorbance (OD) of series of solution of KMnO4 prepared

above by the method described above.

15) Plot a graph between OD against concentration. (If a straight line is

obtained Beer’s law is verified)

16) Now find out the OD of the unknown solution of the KMnO4. Find out the

concentration of this solution from the graph.

OBSERVATION & TABLE:

(i) Determination of ʎmax

Wavelength (nm) Absorbance (OD)

(ii) Verification of Beer’s law

S.NO. Concentration (C) (moles / L) Absorbance (OD)




80

CALCULATION:

(i) A curve is plotted between wavelength and absorbance (OD).

(ii) A curve is plotted between O.D and concentration and if a straight line is

obtained as shown by equation (i), Beer’s law is verified.

(iii) From the graph of O.D versus concentration, the concentration of the

unknown solution can be found out. For example, in the fig x is the O.D of

unknown solution then its concentration will be 1.0%.


(i) ʎmax for KMnO4 = ………nm

(ii) KMnO4 solution obeys Beer’s law

(iii) Concentration of the unknown solution = ………mg/L

PRECAUTIONS:

i) Always use dilute solutions for getting calibration curve.

ii) Cuvette should be cleaned properly and must be wiped with tissue paper.

iii) Do not leave any finger marks on the cuvette.




81

Section: VII

Laboratory DemonstrationsLaboratory DemonstrationsLaboratory DemonstrationsLaboratory Demonstrations

(PCR & ELISA)(PCR & ELISA)(PCR & ELISA)(PCR & ELISA)




82

Experiment No.27

AIM: To Amplify A Specific DNA Fragment By Polymerase Chain Reaction.

INTRODUCTION: PCR is an in vitro method of enzymatic synthesis of specific DNA

fragment developed by Kary Mullis in 1983. It is very simple technique for

characterizing, analyzing & synthesizing DNA from virtually any living organism.

PCR is used to amplify a precise fragment of DNA from a complex mixture of

starting material called as template DNA.

A basic PCR requires the following components:

• DNA template that contains the region to be amplified.

• 2 primers complementary to the 3’ end of each of the sense & antisense strand

of the DNA.

• Thermostable DNA polymerase like Taq, Vent, Pfu etc.

• Deoxynucleotides phosphates (dATP, dCTP, dGTP, dTTP), the building blocks

from which the DNA polymerase synthesizes a new DNA strand.

• Buffer solution which provides a suitable chemical environment for optimal

activity & stability of DNA polymerase.

• Bivalent Mg/Mn ions, which are necessary for maximum Taq polymerase

activity & influence the efficiency of primer to template annealing.

PRINCIPLE: The purpose of a PCR is to amplify a specific DNA or RNA fragment.

PCR comprises of 3 basic steps:

1. Denaturation

2. Annealing

3. Primer extension

� Initialization step: this step consists of heating the reaction mixture to 94-96°C

for 1-9 minutes to break the hydrogen bonds in DNA strands.

� Denaturation step: this step is the first regular cycling event & consists of

heating the reaction mixture to 94-98°C for 20-30 seconds. As a result the




83

template DNA denatures due to disruption of the H-bonds between

complementary bases of the DNA strands, yielding single strand of the DNA.

� Annealing step: in this step the reaction temperature is lowered to 50-65°C for

20-40 seconds allowing annealing of the primer to the ss-DNA template.

Typically the annealing temperature is 3-5°C below the Tm (melting

temperature) of the primers used. Stable DNA-DNA H-bonds are only formed

when the primer sequence very closely matches the template sequence. The

polymerase binds to the primer- template hybrid & begins DNA synthesis.

� Elongation step: in this step the temperature depends on the DNA polymerase

used. Taq polymerase has its optimum activity at 75-80°C. Commonly a

temperature of 68-72°C is used with this enzyme. The DNA polymerase

synthesizes a new strand of DNA, complementary to the DNA template strand

by incorporating dNTPs that are complementary to the template in 5’-3’

direction, condensing the 5’ –phosphate group of the dNTPs with the 3’

hydroxyl group at the end of the nascent DNA strand. The extension time

depends both upon the DNA polymerase used & on the length of DNA

fragment to be amplified. The DNA polymerase will polymerize a thousand

bases per minute at its optimum temperature. Under optimum conditions, i.e.,

if there are no limitations due to limiting substrates or reagents, at each step,

the amount of DNA target is doubled, leading to exponential amplification of

the specific DNA fragments.

� Final elongation: this single step is occasionally performed at a temperature of

70-74°Cfor 5-15min after the last PCR cycle to ensure that any remaining ss-

DNA is fully extended. Denaturation, annealing & extension steps are repeated

20-30 times in an automated thermocycler that can heat & cool the reaction

mixture in tubes within a very short time. This results in exponential

accumulation of specific DNA fragments, ends of which are defined by 5’ ends

of the primers. The doubling of the number of DNA strands corresponding to

the target sequence allows us to estimate the amplification associated with

each cycle using the formula:

Amplification= 2n

, where n= no. of cycles.

� Final hold: this step may be employed for short term storage of the reaction

mixture at 4°C for an indefinite time.




84

MATERIALS REQUIRED:

Glasswares, Ethidium Bromide, Thermocycler, Electrophoretic Apparatus, UV-

Transilluminator, Vortex Mixer, Micropipette & Tips, Crushed Ice etc.

Name of the items Storage

10x assay buffer -20 °C

Control PCR product -20 °C

2.5mM dNTP mix -20 °C

1 kb DNA loader -20 °C

Forward primer (100ng/µl) -20 °C

Reverse primer (100ng/µl) -20 °C

Taq DNA polymerase -20 °C

Template DNA -20 °C

Molecular biology grade water RT

25mM MgCl2 -20 °C

Agarose RT

50X TAE RT

6X gel loading buffer 2-8°C

Mineral oil (optional) RT

PCR tubes RT

PROCEDURE:

1. Preparation of master mix for PCR - To a PCR tube add all the following

ingredients in the following order:

Sr. no. Ingredients Volume in µl

1. Molecular bio. Grade water 31.5

2. 10x assay buffer 5




85

3. Template DNA 1

4. Forward primer (100ng/µl) 1

5. Reverse primer (100ng/µl) 1

6. 25 mM MgCl2 5

7. 2.5 mM dNTP mix 5

8. Taq DNA polymerase 0.5

Total volume 50

2. Tap the tube for 1-2 sec. to mix the contents thoroughly.

3. Add 25 µl of mineral oil in the tube to avoid evaporation of the contents.

4. Place the tube in the thermocycler block & set the program to get DNA

amplification.

Note: it is not essential to add mineral oil if the thermocycler is equipped with a

heating lid.

PCR AMPLIFICATION CYCLE:

Carry out the amplification in a thermocycler for 25-30 cycles using the following

reaction conditions.

Initial denaturation at 94°C for 10 minutes

Denaturation at 94°C for 30 sec

Annealing at 58°C for 30 sec

Extension at 72°C for 45 sec

Final extension at 72°C for 10 min

Cooling at 4°C

25-30 CYCLES




86

AGAROSE GEL ELECTROPHORESIS:

Electrophoresis of the amplified product will be carried out as per the procedure

given in Experiment No.23.

RESULT & INTERPRETATION:




87

Experiment No.28

AIM: To Perform Sandwich ELISA.

INTRODUCTION: ELISA, also called Enzyme Linked Immunosorbent Assay,

employs antigens or antibodies conjugated to enzymes in such a way that the

immunological and enzymatic activity of each component is maintained. These

assays are very sensitive and give accurate results. The estimation of results can

be made either visually or spectrophotometrically. Various formats of ELISA are

available. This method is used for quantitation of antibody.

PRINCIPLE: Sandwich ELISA involves the attachment of a constant dilution of

antibody to the solid phase. After incubation, un-adsorbed antibodies are washed

away. Following that, un-adsorbed reactive sites are blocked on the plate. To that,

antigen at a single dilution or as a dilution range is then added. After incubation

unbound antigen are washed away. Bound antigen is then detected by the

addition of enzyme labeled secondary antibody specific for the "trapped" or

"captured" antigen. After incubation and washing away of unreacted conjugate,

substrate is added and the intensity of the colour is measured.

MATERIALS REQUIRED: ELISA Reader, Distilled water, Glasswares (Conical Flask,

Measuring cylinder, Pipette), Micropipette, Tips

Name of the items storage

Antigen -20ºC

Antibody -20ºC

Sample I -20ºC

Sample II -20ºC

Wash buffer RT

Blocker 4ºC

(dissolve 100mg in 5ml of 1X PBS)

Substrate (freshly prepare) 4ºC

Substrate buffer 4ºC




88

Conjugate 4ºC

Coating buffer 4ºC

Stopping solution RT

Hydrogen peroxide 4ºC

1x PBS RT

ELISA moduls RT

WORKING SOLUTION PREPARATION:

1. Blocking Solution

To prepare 2% blocking solution, take 5ml of 1X PBS and add 100mg of blocker

provided and mix well.

Note: Prepare freshly everytime before each experiment

2. Substrate

With the given substrate quantity, add 1ml of substrate buffer and mix well by

repeat pipetting. To this 1 ml again add 69ml of substrate buffer. Aliquot this

70ml stock solution into 7 separate 10ml storage tubes and wrap it with

aluminium foil and store at -20°C for subsequent usage. This will avoid the loss of

effectiveness of the substrate stock solution at the time of thawing for the

subsequent usage of each test. Take 10ml of the substrate stock solution and mix

with 40µl of hydrogen peroxide. (Prepare this step freshly before each test).

PROCEDURE:

1. Coating Of The Antibody

Dilute the antibody with the coating buffer provided at 1: 100 dilutions

and add 100µl to each well of an ELISA plate. Leave it overnight at 4°C for

passive adsorption of the antibody to the ELISA plates. Use 2 strips/rows

(16 wells) for each test for which you would require 1600µl of diluted

antibody. Make at least 1800 µl of diluted antibody to account for minor

pipetting errors.

2. Washing




89

The concept of ELISA involves separation of bound and free reagents with the

washing step. The un-adsorbed antibody molecules need to be removed by

washing thrice. Washing is done by adding 300 µl of washing buffer to each

well, shaking it mildly and then discarding it vigorously into the sink. This

procedure should be repeat thrice during each washing step. After washing,

flick the plates and dry on a stack of dry filter papers to avoid any bubble

formation that would interfere in subsequent reagent additions.

3. Blocking

After coating and removal of unbound antibody, the remaining sites on the

ELISA plates has to be blocked to avoid direct binding of antigen or conjugate

which would lead to false positive reactions. Hence add 300µl of the blocker

solution to all the wells and incubate the plate at 37°C for 45 minutes.

NOTE: Use higher volumes (300 µl) of blocker for more efficient blocking of the

reactive sites on the sides of the wells also.

4. Washing

After incubation, washing is done as explained earlier.

5. Antigen Addition

After washing the strips, add 1001-11 of the antigen provided at a series of

double dilutions starting from 1:100 to 1: 1600 in phosphate buffered saline (1X

PBS), into the duplicate wells as:




90

Blocking buffer (100µl) can be added to wells F1 and F2 instead of antigen that

would serve as negative control.

Two samples can be used at a dilution of 1: 100 to wells G 1 and G2 and H1 and

H2 to determine the antigen content in those sample using the standard curve

generated from the standards (Wells A to E -1 and 2). Incubate the plate at

37°C for 45 minutes.

6. Washing

After incubation, washing is done as explained earlier.

7. Conjugate incubation

After washing the wells, add 100µl of the diluted conjugate provided (1: 1000 in

phosphate buffered saline) into all the 16 wells of the ELISA plate. Incubate the

plate at 37° for 45 minutes. And then wash.

8. Substrate addition

Add 100µl of the substrate solution into the 16 wells of the plate and incubate

the strips in dark at room temperature for 10minutes.

9. Stop The Reaction And Reading Optical Density

The appearance of yellowish brown colour indicates that the antigen antibody

reaction has occurred. Stop the reaction by adding 25µl of the stop solution.

Read the optical density (OD) values of the plate in an ELISA reader at 490 nm

wavelength.

WELLS 1 2

A ANTIGEN 1:100 ANTIGEN 1: 1 00

B ANTIGEN 1 :200 ANTIGEN 1 :200

C ANTIGEN 1 :400 ANTIGEN 1 :400

D ANTIGEN 1 :800 ANTIGEN 1 :800

E ANTIGEN 1:1600 ANTIGEN 1:1600

F Negative Control Negative Control

G SAMPLE 1 (1:100) SAMPLE 1 (1:100)

H SAMPLE2(1: 100) SAMPLE2(1: 100)




91

RESULTS & INTERPRETETION:

Add 100µl antibody with coating buffer

(incubate for overnight at 4°C)

Add 300 µl of washing buffer

Add 300 µl of blocking solution

(incubate at 37°C for 45min)

Wash

Add 300 µl of antigen

(incubate)

Wash

Add 100 µl of dilute conjugate

(incubate)

Wash

Add100 µl of substrate solution

(incubate)

Observe the result




92

ABOUT THE AUTHORS

Dr Gyanendra Awasthi is presently working as reader and Head, Department of

Biochemistry in Dolphin (P.G.) Institute of Biomedical and Natural Sciences,

Dehradun. He is teaching Biochemistry to UG and PG students since last ten years. He

did his M.Sc. degree in Biochemistry from Lucknow University and Ph.D. from

H.N.B.Garhwal University Srinagar and also qualified CSIR-NET in life sciences .He

has published 2 books more than 20 research papers in national and international

journals of repute.

Dr Santosh Kumar is presently working as Assistant professor, Department of

Biochemistry in Dolphin (P.G.) Institute of Biomedical and Natural Sciences, Dehradun.

He is teaching Biochemistry to UG and PG students since last seven years. He did his

M.VSc. degree in Animal Biochemistry from NDRI; Karnal. He has published more than

05 research papers in national and international journals of repute.

Dr Ashwani Sanghi is presently working as Assistant professor in Department of

Biochemistry in Dolphin (P.G.) Institute of Biomedical and Natural Sciences,

Dehradun. He is teaching Biochemistry to UG and PG students since last 06 years. He

did his M.Sc. degree in Biochemistry and Ph.D. from Kurkushestra University

Kurkushestra. He has published more than 06 research papers in national and

international journals of repute.

Mr Shiv Sharan is presently working as assistant professor Department of Biochemistry

in Dolphin (P.G.) Institute of Biomedical and Natural Sciences, Dehradun. He is teaching

Biochemistry to UG and PG students since last ten years. He did his M.Sc. degree in

Biochemistry from Allahabad Agriculture deemed university Allahabad and pursuing Ph.

D. from Uttrakhand Technical University, Dehradun. He has published more than 05

research papers in national and international journals of repute.

biochemistry laboratory manual - iscaisca.co.in/bio_sci/lab_manual/iep-bs-lm-2013-003.pdf · in the...

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