3.1 reagents and chemicals name sourceshodhganga.inflibnet.ac.in/bitstream/10603/11720/1/10....

MATERIALS AND METHODS

48

3.1 REAGENTS AND CHEMICALS

Name Source

Acetic acid (glacial)

Acetonitrile

Acetone

Acrylamide

Agarose

Ammonium per sulphate (APS)

Ammonium chloride

Ammonium Bicarbonate

Ampicillin

Bacto yeast extract

Bactoagar

Bactotryptone

Bovine serum albumin (BSA)

Bromophenol blue

Calcium chloride

Chloroform

3-[(3-cholamidopropyl)dimethylammonio]-

1-propane sulphonate (CHAPS)

Citric acid

Coomassie brilliant blue R 250 and G250

Copper sulphate

3,3`-Diaminobenzidine (DAB)

Diethylether

Diethyl pyrocarbonate (DEPC)

Diethylene triamine pentaacetic acid

Dithiothreitol (DTT)

5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB)

DNA molecular weight marker

Ethanol

Ethidium bromide (EtBr)

MERCK (India)

MERCK (India)

MERCK (India)

Sigma (U. S. A.)

HiMedia (India)

Sigma (U.S.A.)

MERCK (India)

MERCK (India)

HiMedia (India)

HiMedia (India)

HiMedia (India)

HiMedia (India)

HiMedia (India)

HiMedia (India)

HiMedia (India)

MERCK (India)

Sigma (U.S.A.)

Sigma (U.S.A.)

HiMedia (India)

HiMedia (India)

Sigma (U.S.A.)

MERCK (India)

Sigma (U.S.A.)

HiMedia (India)

Sigma (U.S.A.)

HiMedia (India)

Bangalore genei (India)

MERCK (India)

MERCK (India)


49

Ethylene diamine tetra acetate (EDTA)

Formaldehyde

Glucose

Glutathione (oxidized)

Glycerol

Glycine

Hydrogen peroxide

Igepal CA-630

Imidazole

Iodoacetamide (IAA)

Isopropanol

Isopropyl-thiogalactoside (IPTG)

2-Mercaptoethanol

Methanol

Mineral oil

3-N(Morpholino)propanesulphonicacid

(MOPS)

4-(methylnitrosoamino)-1-(3-pyridyl)-1-

butanone (NNK)

N, N`-methylene bis acrylamide

Nickel-nitriloacetic acid superflow resin

Nickel sulfate

Nicotinamide adenine dinucleotide phosphate

(NADPH)

o-Phenylenediamine (OPD)

Phenol blue

Phosphoric acid

Potassium acetate

Potassium chloride

Potassium di-hydrogen phosphate

Potassium ferricyanide

Protein molecular weight marker

Pyrogallol

HiMedia (India)

MERCK (India)

HiMedia (India)

HiMedia (India)

MERCK (India)

MERCK (India)

MERCK (India)

Sigma (U.S.A.)

MERCK (India)

Sigma (U.S.A.)

MERCK (India)

Bangalore Genei (India)

MERCK (India)

MERCK (India)

BioRad (Hercules, CA)

Sigma (U.S.A.)

Sigma (U.S.A.)

HiMedia (India)

Qiagen (Germany)

MERCK (India)

HiMedia (India)

Sigma (U.S.A.)

HiMedia (India)

MERCK (India)

MERCK (India)

MERCK (India)

MERCK (India)

MERCK (India)

Bangalore Genei (India)

HiMedia (India)


50

Silicone

Silver nitrate

Sodium acetate

Sodium carbonate

Sodium chloride

Sodium dodecyl sulphate (SDS)

Sodium di hydrogen phosphate

di-Sodium hydrogen phosphate

Sodium hydroxide

Sodium bicarbonate

Sodium thiosulphate

Sulphuric acid

Tetramethylethylenediamine (TEMED)

Thiobarbituric acid (TBA)

Thiourea

Trichloroaceticacid (TCA)

Tris (hydroxymethyl) aminomethane

TRIZOL

Trypsin

Tween 20

Urea

HiMedia (India)

MERCK (India)

MERCK (India)

MERCK (India)

MERCK (India)

MERCK (India)

MERCK (India)

MERCK (India)

MERCK (India)

MERCK (India)

MERCK (India)

MERCK (India)

MERCK (India)

HiMedia (India)

Sigma (U.S.A.)

MERCK (India)

SRL (India)

Sigma (U.S.A.)

Sigma (U.S.A.)

MERCK (India)

MERCK (India)

3.2 ANTIBODIES

The serum of confirmed lung cancer patient was kindly obtained from Dr. V.P. Gupta from

Cancer Hospital and Research Institute, Kota, Rajasthan.

Primary antibodies specific for rat and human, such as rabbit polyclonal transferrin, goat

polyclonal apolipoprotein A-I were obtained from Santa Cruz (U.S.A.), chicken polyclonal

alpha1 antitrypsin, and mouse polyclonal IgG1 (heavy chain, constant region) from AbD

Serotec (UK), mouse monoclonal anti His Tag antibody from Genscript (USA)

HRPO conjugated secondary antibodies such as goat anti-rabbit IgG was from AbD Serotec

(UK), rabbit anti-goat IgG, goat anti-chicken IgY were from Abcam (UK) and rabbit anti-

mouse IgG1 was from AbD Serotec (UK), goat anti-mouse IgG from Sigma (USA), rabbit

anti-rat IgG1 from AbD Serotec (UK).


51

3.3 ENZYMES USED FOR CLONING

Restriction enzymes: Bam H1, Hind III and calf intestinal alkaline phosphatase enzyme were

purchased from Fermentas Life Science (Canada).

3.4 PRIMERS AND PCR KIT

cDNA synthesis kit, forward and reverse primers for amplification of 1.4kb gene, PCR

amplification kit and ligation kit were purchased from Bangalore Genei (India). Gel elution kit

or nucleospin extract columns were purchased from Macherey Nagel (Germany).

3.5 HOSTS AND VECTOR USED

Escherichia coli host strains DH5α and BL21 (DE3) for routine recombinant plasmid

manipulations, expression vector pRSETc (ampr) for recombinant protein expression was

obtained from Invitrogen (Carlsbad, U.S.A.).

3.6 OTHER MATERIALS/ITEMS

Albumin depletion kit was procured from Bangalore Genei (India).

Millipore membranes and glass fiber filters were procured from Millipore Co. (U.S.A.).

Nitrocellulose membrane was procured from Schleicher and Schull (Germany).

Immobilized pH 3-10 gradient strips, IPG buffer were procured from GE Amersham (UK).

Sterile tubes, disposable culture dishes, tissue culture flasks, microtips, microfuge tubes and

cryovials were procured from Tarsons (India).

3.7 ANIMALS

Six weeks old pathogen free, male wistar rats were procured from Central Animal House facility

of Jamia Hamdard University, New Delhi. They ranged in weight from 150-180 g. Rats were

housed six per cage and were allowed to acclimatize to the laboratory conditions one week

before the start of experiment under standard conditions (20+/-2oC; 50+/-10% relative humidity;

12 hrs of light /dark cycles). Animals were given free access to modified feed containing high

fat (26.15%) and low protein (6.05%) contents (Table 3.1, Amrut India Ltd., Pune) and water ad

libitum. All the experiments were performed in accordance with the Institutional Animal Ethics

Committee (IAEC) constituted as per directions of the Committee for the Purpose of Control

and Supervision of Experimental Animals (173/CPCSEA) under the Ministry of Animal Welfare

Division, Government of India, New Delhi.


52

Table 3.1 Dietary components of the feed for experimental rats

Sl. No. Components Proximate analysis value (%)

1 Crude Protein 6.05

2 Crude Fat 26.15

3 Crude Fibre 4.10

4 Calcium 1.20

5 Phosphorous 0.55

6 Moisture 8.65

7 Total Ash 9.05

8 Acid insoluble ash 0.75

9 Carbohydrates 43.50

3.8 LUNG CANCER INDUCTION IN ANIMAL MODEL

Lung cancer model was developed in rats by modifying the protocol of Belinsky et al. (1986)

and Morse et al. (1989). Rats were divided into 2 groups (control group and treated group) with

50 animals in each. Rats were injected subcutaneously with a single high dose (2.5 mg/kg body

weight) of NNK followed by repeated reduced doses (1.5 mg/kg body weight) 3 times /week for

total dose of 100 mg/kg body weight. The experiments were terminated after 22 weeks. Control

animals were injected with saline on the same schedule. Five animals were sacrificed at every

two months and lungs, liver and kidneys were taken out for histopathological analyses. Blood

samples from both control and NNK treated rats were also collected and sera separated for

biochemical estimations.

3.9 HISTOLOGICAL EXAMINATION OF RAT LUNG TISSUE

All animals were sacrificed under ether anesthesia and major organs such as lungs, liver and

kidneys of both control and treated rats were quickly removed. The specimens were post-fixed

in buffered formalin (10%) for 24 hr. Following fixation, sections (3-4 mm in thickness) of these

tissues were dehydrated with absolute alcohol, embedded in fresh paraffin and allowed to cool.

At least four cross-sections (5 µm thick) from each tissue were cut on a microtome and stained

with Hematoxylin (H) and Eosin (E). The tissue sections were then washed twice (2 min each)

with xylene and later mounted with DPX mountant. The slides were observed for

histopathological changes and microphotographs were taken using an Olympus BX50

microscope system (Olympus, Japan).


53

3.10 SAMPLE COLLECTION AND SERUM PREPARATION

Rats were anaesthetized with diethyl-ether and blood was drawn through venous puncture.

Blood drawn from both the control and treated rats was allowed to coagulate at RT for 30 min

and then centrifuged at 1200xg for 20 min (Tuck et al., 2009). Serum was then aliquoted and

stored at -80°C until used. Serum was used directly for biochemical estimations while for

proteomic analyses it was depleted of albumin and precipitated by TCA.

3.10.1 Depletion of high-abundant protein (Albumin) from serum

Serum albumin can constitute 60-70% of the total serum protein (Anderson and Anderson,

2002). The concentration of this protein may cause poor resolution of lower abundance proteins

in two-dimensional electrophoresis (2-DE). Depletion of albumin removes approximately 50%

of 75% of total serum proteins, so that 3-4 times more enriched sample can be loaded on 2-DE,

allowing the visualization and analysis of the remaining proteins easier.

Protocol

Column Equilibration

Serum samples from both control and treated rats were processed to selectively deplete albumin.

For this a dye based proteoprep kit (Bangalore Genei) was used according to the manufacturer’s

instructions. The matrix medium is blue dye conjugated on agarose base resin. The medium was

gently resuspended and a uniform suspension was formed by swirling until no settled medium

remained at the bottom of the bottle when inverted. Then 0.5 mL of the suspended medium

slurry was transferred to a spin column. The spin column in the collection tube was spun at

3,000rpm for 30 seconds to remove the storage solution.0.3 mL of the equilibration buffer was

added to the medium in the spin column and it was then centrifuged at 3,000rpm for 30 seconds.

The buffer was then discarded from the collection tube and the spin column was placed back to

the same collection tube. This step was repeated using the same collection tube. The serum

samples (25-100 µl) were then added to the top of the packed medium bed and it was incubated

at RT for 10 min. In this step the sample is immediately adsorbed into the medium ensuring the

efficient binding and minimal sample dilution. The spin columns and the collection tubes were

again centrifuged at 3,000rpm for 30 seconds. The eluted sample was reapplied in the collection

tube at the top of the medium bed. It was incubated at RT for 5 min. This step removes

additional amounts of albumin. The spin columns in the same collection tubes were centrifuged

at 3,000rpm for 30 seconds. Then the remaining unbound protein was washed from the spin


54

column by adding 200 µl of equilibration buffer (1X) to the top of the medium bed and

centrifuging it at 3,000rpm for 30 seconds. The depleted serum samples were recovered and

stored at -80oC for long term storage.

3.10.2 TCA-acetone precipitation

Contaminants in protein extracts may include a variety of macromolecules (nucleic acids, lipid

micelles, polysaccharides, and other proteins) as well as an assortment of small molecules.

Small molecules are fairly easy to separate from proteins by size selection such as dialysis,

ultrafiltration, and gel filtration. In the process of precipitating proteins with salts or other

agents, small molecules usually stay in solution. Effective separation is then accomplished by

centrifugation, causing precipitated macromolecules to collect as a pellet at the bottom of the

centrifuge tube. Macromolecules are more difficult to remove, especially when present in large

amounts. So the most suitable method should be employed for sample preparation.

Precipitation is widely used in downstream processing of biological products, such as proteins

because it can concentrate samples, remove salts, and polysaccharides, and denature endogenous

proteases (Pridmore et al., 1999, Jacobs et al., 2001). Proteins can be efficiently precipitated

with trichloroacetic acid (TCA), acetone, or even ethanol, although the concentrations at which

these (mostly) miscible organic solvents function can vary greatly. As with ammonium sulfate,

the mechanism of precipitation is hydrophobic aggregation. However, in addition to disruption

of the solvation layers of the proteins, these compounds also partially denature the proteins,

exposing even more hydrophobic surface to the solvent.

In practical, TCA is often the compound of choice amongst the miscible organics (Berkelman

and Stenstedt, 1998; Molloy et al., 1998) because it is effective at lower concentrations than the

others – ~15% for TCA, ~75% for acetone, and ~90% for ethanol – which means the sample

volume doesn’t increase dramatically. Because of this, the protein concentration remains higher

during a TCA precipitation, increasing the efficiency of the precipitation. But because TCA is an

acid, the protein pellet is either washed with 75% acetone to remove the TCA, or base is added

after the pellet is resuspended in SDS-PAGE sample buffer (until the bromophenol blue turns

from yellow back to blue) to neutralize the pH. TCA-Acetone precipitation is also

experimentally shown to improve the resolution of the protein profile generated during 2-DE

(Ahmad et al., 2009).

http://en.wikipedia.org/wiki/Downstream_processing

http://en.wikipedia.org/wiki/Proteins


55

Protocol

A 100 μl of albumin depleted serum sample was diluted with 900 μl of 10% TCA in acetone.

The mixture was incubated overnight at -20°C and centrifuged at 15,000xg, 4°C for 10 min. The

supernatant was removed and 1000 μl of 90% ice-cold acetone were added to wash the pellet.

The sample was incubated at -20°C for 10 min and centrifuged as above. The acetone containing

supernatant was removed and the pellet was air dried. For 2D gel electrophoresis, the protein

pellet was suspended in 100 μl of lysis buffer containing 8 M urea, 2.5 M thiourea, 40 mM Tris-

HCl, 3% w/v CHAPS and 0.5 % v/v Igepal CA-630. The protein sample was stored frozen at -

80°C until analysis.

3.10.3 Protein estimation

The protein content in sera was estimated by the method of Bradford (1976) using bovine serum

albumin as standard.

Principle

The Bradford assay is based on the observation that the absorbance maximum for an acidic

solution of coomassie brilliant blue G-250 shifts from 465 nm to 595 nm when binding to

protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of dye

causing a visible color change.

Solutions

Bradford Reagent (1 L)

Components Amount

Ethanol (95%) 50 mL

Phosphoric acid (85%) 100 mL

Coomassie brilliant blue dye (G-250) 100 mg

Water Upto 1 litre

The mixture was dissolved well and filtered through Whatman # 1 paper just before

use. The bradford reagent should be light brown in color. Therefore filtration was

repeated to rid the reagent of blue components.


56

Bovine Serum Albumin (BSA, 100 mL)

Components Amount

BSA 1 g

Tris-HCl (10 mM, pH=7.5) 100 mL

Protocol

Spectrophotometer was warmed up before use. Unknown proteins were diluted to 100 µl with

water. Standards were prepared containing a range of 5 to 100 µg protein (10 mg/mL BSA) in

100 µl volume. Then 900 µl of dye reagent was added and allowed to incubate for 5 min and

absorbance was measured at 595 nm. A standard curve of absorbance versus microgram of

protein was prepared and protein concentration was determined from the curve.

3.11 BIOCHEMICAL MEASUREMENTS FROM RAT SERA

Biochemical analyses from the sera obtained from control as well as treated group of animals

were performed according to the methods described below. The animals were sacrificed after

every two months and were systematically dissected to take out the lungs. At this stage blood

samples were collected and sera at 1, 3, 5, 7 and 9 months were analysed for biochemical

estimations in both NNK treated and controls.

3.11.1 Estimation of lipid peroxidation

Thiobarbituric acid reactive substances (TBARS), malondialdehyde (MDA) is a measure of the

end product of lipid peroxidation. It was determined by the method as described by Wright et al.

(1981). Reaction mixture in a total volume of 2.0 mL contained 1.8 mL of phosphate buffer (0.1

M, pH 7.4), 0.2 mL of serum. The reaction mixture was incubated at 37°C in a shaking water

bath for 1 hr. The reaction was stopped by the addition of 1.0 mL trichloroacetic acid (TCA)

(10% w/v). Following the addition of 1.0 mL thiobarbituric acid (TBA) (0.67% w/v, prepared in

warm distilled water) all the tubes were placed in a boiling water bath for 20 min. In the end,

tubes were cooled on ice and centrifuged at 2,500xg for 10 min, the supernatant containing the

thiobarbituric acid reactive substances (TBA-RS) formed in each sample was assessed by

measuring the optical density at 535 nm. The results were expressed as nmol of

malondialdehyde (MDA) formed/mg protein using a molar extinction coefficient of 1.56×105/ M

/cm.


57

3.11.2 Assay of enzymatic antioxidants

3.11.2 (a) Estimation of superoxide dismutase (SOD) activity

Superoxide dismutase activity was determined for its ability to inhibit the auto-oxidation of

pyrogallol according to the method of Nandi and Chatterjee (1988). The reaction mixture (1 mL)

consisted of 50 mM Tris (hydroxymethyl) aminomethane (pH 8.2), 1 mM diethylenetriamine

penta acetic acid, and 50 µl of serum sample. The reaction was initiated by the addition of

pyrogallol (final concentration of 0.2 mM), and the absorbance was measured kinetically at 420

nm (25ºC) for 3 min. SOD activity was expressed as unit/mg protein.

3.11.2 (b) Estimation of catalase (CAT) activity

Catalase is a ubiquitous antioxidant enzyme that is present in nearly all living organisms. It

functions to catalyze the decomposition of hydrogen peroxide (H₂O₂) to water and oxygen. Its

activity was determined by the method of Claiborne et al. (1985). The assay mixture consisted

of 1.8 mL phosphate buffer (0.05 M, pH 7.4), 1.0 mL hydrogen peroxide (0.019 M) and 0.2 mL

serum in a total volume of 3.0 mL. The rate of change of absorbance per minute at 240 nm was

recorded and catalase activity was expressed as µ mol H2O2 decomposed/min/mg protein using

extinction coefficient of 0.081 x 103/ M/cm.

3.11.2(c) Estimation of glutathione reductase (GR) activity

Glutathione reductase activity is measured by monitoring the amount of reduced nicotinamide

adenine dinucleotide phosphate (NADPH) consumed in the conversion of oxidized glutathione

(GSSG) to reduced glutathione (GSH) in a GR catalysed reaction. GR was assayed by the

method of Mohandas et al. (1984). The assay system taken in a 3 mL quartz cuvette consisted of

1.73 mL phosphate buffer (0.1 M, pH 7.4), 0.1 mL NADPH (0.1 mM, prepared fresh), 0.1 mL of

EDTA (0.5 mM), 0.05 mL of oxidized glutathione (1 mM) and 20 µl serum sample in a total

volume of 2.0 mL. The enzyme activity was quantified by measuring the disappearance of

NADPH at 340 nm at 30 sec intervals for 3 min. The activity was calculated using a molar

extinction coefficient of 6.22×103/M/cm and was expressed as nmol NADPH oxidized/min/mg

protein.

3.11.3 Assay of non-enzymatic antioxidant

3.11.3 (a) Estimation of reduced glutathione (GSH)

Reduced glutathione in serum was determined by modifying the method of Moron et al. (1979).

The assay involves oxidation of GSH by the sulfhydryl reagent 5,5′-dithio-bis(2-nitrobenzoic


58

acid) (DTNB) to form the yellow derivative 5′-thio-2-nitrobenzoic acid (TNB). 1 mL of serum

was precipitated with 1 mL of trichloroacetic acid (10% w/v). The samples were kept at 4°C for

1hr, centrifuged at 2000xg for 20 min at 4°C and the supernatant was separated. The assay

mixture in a total volume of 3.0 mL contained 0.1 mL of above supernatant, 2.7 mL phosphate

buffer (0.1 M, pH 7.4) and 0.2 mL DTNB (100 mM). The yellow color developed was read

immediately at 412 nm. The levels of GSH were expressed as µg/mg protein.

Statistical analysis

The data are expressed as Mean +/- Standard Deviation. Analyses are done using GraphPad

Instat 3 software. Comparisons of the means of sera of control and treated rats were made by

repeated measures ANOVA followed by Tukey test, with, P< 0.05 (*), P<0.01(**) and P<

0.001(***) as limits of significance.

3.12 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) OF RAT AND

HUMAN SERA SAMPLES

Principle

SDS-PAGE describes a technique in molecular biology to separate proteins according to their

electrophoretic mobility (a function of the length of a polypeptide chain and its charge). In most

proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per

unit mass, thereby resulting in a fractionation by approximate size during electrophoresis.

Stock reagents and buffers

Acrylamide mixture (100 mL)

Components Amount

Acrylamide 30% (w/v)

Bis-acrylamide 0.8% (w/v)

Water Upto 100 mL

It should preferably be prepared fresh each time of use. It should be noted

that acrylamide has neurotoxic property and therefore should be used

with utmost precaution. Gloves should be worn during its handling.


59

Resolving and Stacking Buffers (50 mL)

Components (50 mL) Amount

Resolving Buffer (1.5 M Tris-HCl, pH=8.8) 9.09 g

Stacking Buffer (1 M Tris-HCl, pH=6.8 6.06 g

Water Upto 50 mL

Tracking dye (10 mL)

Components Amount

Glycerol 5.8 mL

Phenol blue dye 5 mg

Water Upto 10 mL

Filtered and stored at 4°C

Running buffer (pH=8.3, 1 L)

Components Amount

SDS 1 g

Glycine 14.4 g

Tris 3 g

Water Upto 1000 mL

Sample buffer (50 mL)

Components Amount

2% SDS 1 g

10% Glycerol 5 mL

0.625 M Tris-HCl (pH=6.8) 3.78 g

0.05% Bromophenol blue 3 g

5% 2- mercaptoethanol 2.5 mL

Water Upto 50 mL

2-mercaptomethanol has toxic properties and so should be used precautiously


60

Composition of SDS PAGE gels

Reagents Resolving gel

(12%,40 mL)

Resolving gel

(15%, 40 mL)

Stacking gel

(5%,5 mL)

Water 13.2 9.2 3.4

30% Acrylamide Mixture 16 20 0.85

1.5 M Tris-HCl (pH=8.8) 10 10 -

1 M Tris-HCl (pH-6.8) - - 0.63

10% SDS 0.40 0.4 0.05

10% APS 0.40 0.4 0.05

TEMED 0.025 0.020 0.005

Protocol

Gradient SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on 12%

separating gel with the discontinuous buffer system as described by Laemmli (1970) to see the

protein profiles of albumin depleted and TCA precipitated (A) sera samples from control and

NNK treated rats (B) sera samples from healthy human and lung cancer patient. Samples for

electrophoresis were solublized in sample buffer with 2-mercaptoethanol at 100oC for 3 min.

Electrophoresis was performed on 19cm x 15cm analytical polyacrylamide gels of 1.5 mm

thickness in vertical electrophoresis apparatus (Scientific Systems, New Delhi, India). The

system was connected to the power supply (E-300, Pharmacia, Sweden) and the gel was pre-run

for 15 min at 20 V. Equal amount of protein samples (40-80 µg) were loaded into the wells and

electrophoresis was carried out at a constant current of 30 mA until the dye front reached the

bottom of the gel.

3.13 SILVER STAINING

Principle

In silver staining, polyacrylamide gels are impregnated with soluble silver ion (Ag+) and

developed by treatment with a reductant. Macromolecules in the gel promote the reduction of

silver ion to metallic silver (Ag0), which is insoluble, and visible, allowing protein or nucleic

acid-containing bands to be seen. The initial deposition of metallic silver promotes further

deposition in an autocatalytic process, resulting in exceptionally high sensitivity.


61

Solutions

Reagents (300 mL) Components

Fixing solution 50%Methanol, 5% Glacial Acetic Acid, 45% Methanol

Sensitizing solution 0.02% sodium thiosulphate

Staining solution (stored at 4°C

in amber bottle)

0.1 % silver nitrate

Developing solution 2% Sodium carbonate + 0.08% formaldehyde

Stopping solution 5% acetic acid

Formaldehyde is toxic, and should be handled under a fume hood.

Destaining solution (300 mL)

Components Amount

Sodium thiosulphate 7.44 g

Potassium ferricyanide 2.97 g

Water Upto 300 mL

Method: Incubate gel or gel pieces in destaining solution with gentle shaking,

approximately for 2-3 min or till protein bands become invisible. The gel becomes

yellow. Wash the gel 4-5 times with plenty of milli-Q water until the gel becomes

transparent and has no background.

Note that water used for preparing above stock reagents, buffers and gels should be Milli-Q

water.

Protocol

The gels were incubated in the fixing solution (50% Methanol, 5% Glacial Acetic Acid) for 1-2

hr and were then transferred to 50% methanol solution for 10 min. After washing the gels with

distilled water the gels were incubated with constant shaking for 2 min in 0.02% sensitizing

solution containing sodium thiosulphate. The gels after incubation were rinsed twice with

distilled water for 1 min each. The gels were later stained in 0.1% silver nitrate solution (chilled)

for 20 min with constant shaking. The solution was discarded and gels were rinsed gently with

distilled water for 5 min. For visualization of the protein bands, the gels were put into a freshly

prepared developing solution containing 2% sodium carbonate and 0.08% formaldehyde.

Reaction was stopped by adding 5% glacial acetic acid to the gels after the desirable intensity of

the protein bands was obtained.


62

Determination of molecular weights

The molecular weight of each protein band was determined by calculating the relative mobility

(Rf) value for each of the band. Rf value of a protein band is described as the ratio of the

distance (in cm) migrated by that band in a gel and the total length of the run (distance migrated

by the dye front of the sample). In one of the lanes, the standard molecular weight markers

(proteins of known mol wt) were run and the Rf value of each marker protein was plotted

against the log of its molecular weight. The respective molecular weights of each protein band in

total serum sample were calculated by comparing their Rf values against the standard curve.

3.14 TWO-DIMENSIONAL GEL ELECTROPHORESIS OF RAT SERA SAMPLES

Principle

2D electrophoresis is a powerful technique and considered to be the best option for high-

resolution profiling of low abundance proteins. This technique sorts proteins according to two

independent properties in two discrete steps: the first-dimension step, isoelectric focusing (IEF),

separates proteins according to their isoelectric points (pI); the second-dimension step, SDS-

polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins according to their

molecular weights (Mr, relative molecular weight). For resolution of the proteins of similar

molecular sizes or with very little difference in molecular weights, serum proteins were

subjected to 2D-electrophoresis using O`Farrell method (1975).

Stock reagents and buffers

Rehydration buffer (25 mL)

Components Amount

Urea 8 M

CHAPS 2%

IPG buffer 0.5%

DTT 30 mM

Bromophenol blue 0.002%

Water Upto 25 mL


63

Equilibration buffer I (200 mL)

Components Amount

Urea 6 M

Tris-HCl (pH=8.8) 75 mM

Glycerol 30%

SDS 2%

DTT 2%

Bromophenol blue 1%

Water Upto 200 mL

Equilibration buffer II (200 mL)

Components Amount

Urea 6 M

Tris-HCl (pH=8.8) 75 mM

Glycerol 30%

SDS 2%

Iodoacetamide (IAA) 2.5%

Bromophenol blue 1%

Water Upto 200 mL

Sample buffer (5 mL)

Components Amount

Urea (8 M) 2.4 g

Thiourea (2.5 M) 0.95 g

Tris (40 mM) 0.024 g

CHAPS (4%) 0.2 g

Igepal CA-630 (0.5%) 25 µl

Water Upto 5 mL

Running buffer (pH=8.3, 1 L)

Prepared as mentioned in section 3.7


64

Protocol

First dimension: Isoelectric Focusing (IEF)

Rehydration and sample application

In the first dimension, proteins were separated by isoelectric focusing using immobolized linear

pH gradient (IPG) strips (pH 3-10, 11 cms). 100-120 µg of total serum protein sample (albumin

depleted and TCA precipitated) diluted in rehydration buffer was loaded to obtain a total volume

of 200 µl per IPG strip. The strip was allowed to rehydrate to its original thickness (0.5 mm)

overnight at RT for 12-16 hr. Rehydrated IPG strips were then focused using Ettan IPG phor II

isoelectric focusing system (GE Amersham) according to following protocol:

500 V--------1 hr, 1000 V (Gradient) ------1 hr, 6000 V (Gradient) ------2 hr, 6000 V for a total

of 40,000 Vhrs

After IEF, IPG strips were washed with MilliQ water and incubated in 10 mL of equilibration

buffer I on rocking bed for 15 min, followed by 15 min incubation in the equilibration buffer II

on rocker.

Second dimension: SDS-PAGE

The IPG strip was then soaked in running buffer and placed on top of a 12-15% SDS-

polyacrylamide gel. Electrophoresis of proteins was carried out at RT at 100 V for 6 hr using

Midi-Protean 3 cell (Bio-Rad, Hercules, USA) along with a protein marker. For visualizing the

resolved spots, the gels were silver stained as described in sections above.

Image analyses

Gel images were scanned with image scanner (Biorad Versa doc 4000 MP). Spot detection,

quantification, matching and comparison of 2DE protein patterns of both control and treated

serum samples were done with PD Quest 2D analysis software version Advanced 8.0 (Biorad,

Hercules, CA, USA). PD Quest is a software package for imaging, analyzing and data basing

raw 2-D electrophoresis gels. It runs on windows or Macintosh and has a graphical interface

with standard pull-down menus, toolbars and keyboard commands. For protein matching serum

protein gel maps of normal controls were regarded as referenced gel, artificial matched spots

were built up and the analysis of differential expression was performed by applied software. The


65

spots that were either present in only one of the groups (control vs treated) or showed

statistically significant changes in expression intensity were selected for LC-MS/MS analysis.

In-gel tryptic digestion

The chosen protein spots in 2DE maps were excised manually using a clean ethanol wiped

scalpel followed by washing thrice with 1 mL milliQ water for 10 min each. Next the spots were

destained by adding 15 mM potassium ferricyanide and 50 mM sodium thiosulphate for 5-10

min until the gel pieces start to appear yellow in color. The supernatant was discarded and 250

µl of 200 mM ammonium bicarbonate was added for 15 min until the gel pieces turn colorless.

200 µl of 50:50 (v/v) 200 mM ammonium bicarbonate: acetonitrile was added to the gel pieces

kept on rocker for 15-20 min. Further supernatant was discarded and the gel pieces were

dehydrated with HPLC grade acetonitrile. On complete evaporation of acetonitrile, each gel

piece was rehydrated at 4oC in 20 ng/µl trypsin prepared in 50 mM ammonium bicarbonate. The

gel pieces were incubated overnight at 37oC. 1% formic acid was added to the tubes to stop the

action of trypsin. The sample was spinned down and the supernatant containing tryptic peptides

was collected and stored at -20oC until ready for analysis.

3.15 PROTEIN IDENTIFICATION IN RAT SERA BY LIQUID CHROMATOGRAPHY

TANDEM-MASS SPECTROMETRY (LC-MS/MS)

Principle

Liquid chromatography–mass spectrometry (LC-MS/MS) is an analytical chemistry technique

that combines the physical separation capabilities of liquid chromatography with the mass

analysis capabilities of mass spectrometry. LC-MS/MS is a powerful technique used for many

applications which has very high sensitivity and selectivity. Generally its application is oriented

towards the general detection and potential identification of chemicals or proteins from a

complex mixture by determining their molecular weights and structures. Liquid chromatography

coupled with tandem mass spectrometry is more sensitive compared to LC-MS in a multi-

targeted screening approach.

Protocol

The nano LC was performed with an Agilent 1100 NanoLC-1100 system combined with a

microwell-plate sampler and thermostated column compartment for pre-concentration (LC

http://en.wikipedia.org/wiki/Analytical_chemistry

http://en.wikipedia.org/wiki/High_performance_liquid_chromatography

http://en.wikipedia.org/wiki/Mass_spectrometry


66

Packings, Agilent). Approximately 6 µl of the sample was loaded on the column (Zorbax

300SB-C18, 150 mm X 75 um, 3.5 um) using a pre-concentration step in a micro pre-column

cartridge (Zorbax 300SB-C18, 5 mm X 300 um, 5 um) at a flow rate of 5 µl/min. After 5 min,

the pre-column was connected with the separating column, and multistep gradient (3% till 5min,

15% for 5-8 min, 45% for 8-50 min, 90% for 50-55 min, 90% for 55-70 min, then again 3% for

71 min) was started. The buffers used were 0.1% formaldehyde in water (A) and 0.1%

formaldehyde in 90% ACN (B). Calibration of the instrument was performed using the standard

Agilent tune mix. An LC/MSD Trap XCT with a nano-electrospary interface (Agilent) operated

in the positive ion mode was used for MS. Ionization (1.5 kV ionization potential) was

performed with a liquid junction and a non-coated capillary probe (New Objective, Cambridge,

USA). Peptides get ionized in the liquid phase in the Electrospray ionizer and enter the ion trap,

get fragmented (MS/MS) and detected there. Peptide ions were analyzed by the data-dependent

method as follows: The scan sequence consisted of 1 full MS scan followed by 4MS/MS scans

of the most abundant ions. Each MS/MS spectrum (corresponding to a specific peptide

sequence) generated was then used to search for matched peptides with the help of MASCOT

software (Matrix Science) using Agilent Ion Trap Analysis software version 5.2. Next the

National Center for Biotechnology non-redundant (NCBInr) database was searched with rat as

the taxonomy. A protein hit was identified often by multiple independently sequenced peptides

from the same protein and the data were checked for consistent error distribution. In Mascot, the

ion score for an MS/MS match defined as -10log(P) is based on the calculated probability, P,

that the observed match between experimental data and the database sequence is a random

event. Scores >39 were considered significant (P<0.05). All protein identifications were in the

expected size range based on the position in the gel.

3.16 DETECTION OF PROTEINS (SELECTED AFTER LC-MS/MS) IN RAT SERA BY

IMMUNOBLOT ANALYSIS

To confirm the presence/ absence of four proteins (selected after LC-MS/MS analysis) in sera of

rats and to study their expression pattern with disease progression, western blotting was carried

out with specific antibodies.

Principle

The protein bands separated as the result of SDS-PAGE can be further analyzed to determine

their antigenic properties by using western blotting. The first step in this technique is the transfer


67

of separated protein bands from the gel onto a PVDF/nitrocellulose membrane by

electrophoresis (electro blotting). In electro blotting a sandwich of gel and nitrocellulose

membrane is compressed in a cassette and immersed, in a blot buffer, between two parallel

electrodes. The current is passed at right angles to the gel which causes the separated protein

bands to electrophorese out of the gel, on to the membrane. The membrane with transferred

protein bands is referred as blot. The further analysis of separated protein bands can be done by

probing them with specific primary antibodies detected with labeled secondary antibody.

Chemicals

Transfer Buffer (1X, 1 L, pH=8.3)

Components Amount

Glycine 2.9 g

Tris-HCl 5.8 g

SDS 0.3 g

Methanol 200 mL

Water Upto 800 mL

Washing Buffer; Tris buffer saline-Tween 20 (TBST, 1X, 1 L, pH=7.4)

Components Amount

NaCl 8 g

KCl 2 g

Tris-HCl 3 g

Tween-20 (0.1%) 1 mL

DAB solution

Components Amount

3,3`-diaminobenzidine

(DAB)

15 mg

TBS, (pH= 7.4) 50 mL

30% H2O2 15 μl

Mixed well and used immediately.


68

Protocol

Western blotting was carried out by Towbin method (Towbin et al., 1979). To confirm the

results of LC-MS/MS, an equal amount of rat serum protein (25-30 μg) was loaded and

separated by 10% denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After

the proteins were fractionated, the gel was taken out of the sandwich by prying the glass plates

apart. Four pieces of Whatman 3 mm filter paper and one piece of nitrocellulose filter were cut

to the exact size of the polyacrylamide gel. The nitrocellulose filter was allowed to float on the

surface of a tray containing distilled water to wet it by capillary action. Four pieces of the filter

paper were also soaked in a small amount of transfer buffer. The transfer apparatus was then set

up by placing one 3 mm sponge on the opened, submerged cassette. It was followed by two

pieces of presoaked filter paper, the nitrocellulose membrane, the SDS-polyacrylamide gel, two

more pieces of filter paper and finally the 3 mm sponge. A glass pipette or a test tube was gently

rolled over the assembly to remove any trapped air bubbles. The cassette was closed and pressed

lightly to lock the tabs. The HoeferR

Transfer western blotting apparatus (Pharmacia Biotech,

California, USA) was filled with transfer buffer and the gel-cassette was placed in it, with the

nitrocellulose membrane facing the cathode and the gel facing towards the anode. The transfer

was carried out at 4°C at a constant voltage of 100 V for 2-3 hr.

After the transfer was complete, the cassette was taken out of the tank and opened. The sponge,

filter papers and the gel were removed while the nitrocellulose filter was placed in a tray with

blocking solution containing 3% BSA (Bovine Serum Albumin) and 0.1% Tween 20 in Tris-

buffered saline (TBS) for 1 hr at RT. The membrane was then incubated with rat specific

primary antibodies [rabbit polyclonal transferrin, goat polyclonal apolipoprotein A-I; 1:500,

chicken polyclonal alpha1 antitrypsin and mouse polyclonal IgG1 (heavy chain, constant

region); 1:1000] in blocking solution at 4°C overnight, with shaking. The membrane was rinsed

thrice with 0.1% Tween 20 in TBS and incubated with horse radish peroxidase-conjugated

secondary antibodies (goat anti-rabbit IgG; 1:5,000, rabbit anti-goat IgG, goat antichicken IgY;

1:10,000 and rabbit anti-mouse IgG1; 1:5,000) for 1 hr at RT, with shaking. The membrane was

again washed with 0.1% Tween 20 in TBS three times and the immunoreaction was detected

using 10 mL of DAB (3,3-diaminobenzidine tetrahydrochloride) solution in 1X TBS buffer and

30% H2O2 and stopped when the desired intensity was obtained.


69

3.17 VALIDATION OF PROTEINS (SELECTED AFTER LC-MS/MS) IN HUMAN

SERA BY (A) WESTERN BLOT ANALYSIS (B) ENZYME LINKED

IMMUNOSORBANT ASSAY (ELISA)

(A) Western blot

To validate the expression levels of the Immunoglobulin gamma1 (heavy chain) in human sera,

western blot analysis was carried out with specific antibodies as described in section 3.16.

Primary antibody used was human specific mouse polyclonal IgG1 [(heavy chain, constant

region)]; 1:1000 and secondary antibody used was HRPO-conjugated rabbit anti-mouse IgG1;

1:5,000. The immunoreaction was detected using 10 mL of DAB (3,3-diaminobenzidine

tetrahydrochloride) solution in 1X TBS buffer and 30% H2O2 and stopped when the desired

intensity was obtained.

(B) ELISA

To confirm the presence/ absence of three proteins (transferrin, apolipoprotein A-I and serum

alpha1 antitrypsin, selected after LC-MS/MS analysis) in sera of humans, ELISA assay was

carried out with specific antibodies.

Principle

As a "wet lab" analytic biochemistry assay, ELISA involves detection of an "analyte" (i.e. the

specific substance whose presence is being quantitatively or qualitatively analyzed) in a liquid

sample by a controlled sequence of biochemical reactions that will generate a signal which can

be easily measured quantified and interpreted as a measure of the amount of analyte in the

sample.

Chemicals

Coating Buffer (50 mM, 100 mL, pH=9.6)

Reagents Amount

Sodium carbonate

(Na2CO3)

0.158 g

Sodium bicarbonate

(NaHCO3)

0.284 g

Water Upto 100 mL


70

Citrate Phosphate Buffer (CPB)

Reagents Amount

Solution A (Citric acid) 2.10 g/100 mL

Solution B (sodium di-hydrogen phosphate;

NaH2PO4)

2.84 g/100 mL

Mix Solution A (9.7 mL) and Solution B (10.3 mL) for 20 mL CPB and add 20 mg o-

phenylenediamine (OPD) and 60 µl H2O2 (at the time of use)

Washing Buffer; Phosphate buffer saline-Tween 20(1X PBS-T; 1 L, pH=7.5)

Reagents Amount

Sodium chloride (NaCl) 8 g

Potassium chloride (KCl) 0.2 g

di-sodium hydrogen phosphate

(Na2HPO4)

1.44 g

di-potassium hydrogen phosphate

(KH2PO4)

0.24 g

Tween-20 0.05%

Water Upto 1000 mL

Blocking Solution: 3% BSA in PBS-T

Stopping Solution: 5 N Sulphuric acid (H2SO4)

Protocol

ELISA was carried out according to the protocol of Engvall and Perlman (1971). 96-well ELISA

plates were coated (in duplicate) with desired concentration (3 μg) of human sera (from healthy

human and lung cancer patient) containing the particular antigens/proteins (prepared in coating

buffer, pH 9.6) to a final volume of 100 μl per well. The plates were incubated overnight at 4˚C.

Next day the wells were washed with 1X PBS-T buffer. The non-specific sites were blocked

with 150 µl of 3% BSA. The plates were incubated at 37˚C for 1 h, 30 min. The wells were

washed twice with 1X PBST. Human specific primary antibodies (rabbit polyclonal transferrin,

goat polyclonal apolipoprotein A-I, chicken polyclonal alpha1 antitrypsin) were prepared in PBS

in various dilutions ranging from 1:50 to 1:3000 and 150 μl of each dilution was added to the


71

wells. The plates were further incubated at 37˚C for 1 h. The wells were washed thoroughly with

1X PBST. Secondary antibodies (goat anti-rabbit IgG, Rabbit anti-goat IgG, goat antichicken

IgY diluted at 1:10,000 in 1X PBS buffer) were added to a final volume of 100 μl per well. The

plates were incubated at 37˚C for 1 h. The wells were washed thoroughly with 1X PBST (three

times). Next, 200 μl of the substrate, ortho-phenyl-diamine solution (OPD) was added to each

well. The plates were incubated at 37˚C for 10 min. The reaction was stopped by addition of 50

μl of 5 N H2SO4. The absorbance was recorded at 490 nm.

3.18 THE cDNA SYNTHESIS AND CLONING OF IMMUNOGLOBULIN GAMMA1

HEAVY CHAIN (1.4 kb)

3.18.1 RNA ISOLATION

Reagents

DEPC (Diethyl pyrocarbonate) water

0.1% DEPC dissolved in MilliQ water. Stir it overnight and autoclave.

MOPS electrophoresis buffer (10X, 500 mL)

Components Amount

3-N(Morpholino)propanesulphonicacid

(MOPS)

20.4 g (pH=7.0)

Sodium acetate 3.4 g

EDTA 1.9 g

Water Upto 500 mL

Adjust pH with KOH and autoclave.

Gel loading dye (1X)

Components Amount

Deionized formamide 10 mL

0.5 M EDTA (pH 8.0) 200 µl

A pinch of bromophenol blue was added to get faint blue color

and stored at 4°C for future use.


72

Ethidium bromide (10 mg/mL)

Components Amount

EtBr 0.1 g

Distilled water 10 mL

Prepared the solution in a dark bottle and stored at RT.

EDTA (0.5 M)

Components Amount

Na2EDTA.2H2O 186.1 g


Stirred vigorously on a magnetic stirrer. Adjusted the pH to 8.0 with

NaOH. Dispensed into aliquots and sterilized by autoclaving.

Protocol

Total RNA from NNK treated rat lungs was isolated by the method described by Chomczynski

and Sacchi (1987). Tissue samples (100 mg) were homogenized in Trizol reagent (1 mL) and

then allowed to stand at RT for 30 min. This ensures complete dissociation of nucleoprotein

complex. Then 0.2 mL of chloroform was added to the samples, shaken vigorously for 15

seconds and allowed to stand for 15 min at RT. Resulting mixture was centrifuged for 15 min, at

13,000rpm, at 4oC. Centrifugation separated the mixture into 3 phases: a red organic phase

containing protein, an interphase containing DNA, and a colorless upper aqueous phase

containing RNA. RNA was transferred to a fresh tube and 0.5 mL of isopropanol was added to

it. Sample was allowed to stand for 30 min at -20oC and later centrifuged for 10 min at

13,000rpm, 4oC. Supernatant was discarded and RNA pellet obtained at the bottom and side of

the tube was washed with 1 mL of 70 % ethanol. Sample was vortexed and centrifuged for 5 min

at 13,000rpm, 4oC. RNA was air dried for 5-10 min and then dissolved in appropriate volume of

nuclease free water. Finally RNA sample was stored at -80oC until evaluation.

3.18.2 Agarose gel electrophoresis of RNA

1.2% denaturing gel was used for analysis of RNA. The appropriate amount of agarose was

added to a flask with 1X MOPS buffer and boiled. The solution was cooled and ethidium

bromide (0.5 μg/mL) and 700 μl formaldehyde was added and the mixture was poured into a

casting tray containing the sample comb and allowed to solidify. After the gel had solidified, the


73

comb was removed carefully and the gel was mounted onto the electrophoresis chamber and

covered with 1X MOPS buffer. RNA samples (2 µg) were mixed with 5X formaldehyde running

buffer, 6Xgel loading dye and then incubated at 65oC for 15 min and chilled on ice. The

samples were centrifuged briefly, loaded into the wells of the submerged gel and was allowed to

run at a constant voltage of 80 V. After the gel was run ¾ of the length of the tray, it was

visualized on UV transilluminator.

3.19 PRIMER DESIGNING

The amino-terminal sequence of the 55kDa protein (Immunoglobulin gamma1 heavy chain,

constant region) as determined by LC-MS/MS analysis was subjected to NCBI BLAST to

deduce the primary nucleotide sequence of the gene encoding the protein. The availability of the

sequence data for the protein facilitates the design of oligonucleotide primers for the isolation

and characterization of the corresponding gene. The 31 base oligonucleotide primers were

designed for amplification of 1.4kb gene corresponding to 52.4kDa protein. Restriction analysis

of the gene was performed by submitting the gene sequence in Webcutter DNA program

(Webcutter @ Carolina Biological Supply Company). The restriction sites of enzymes Bam H1

and Hind III respectively were introduced in the forward and reverse primers, which do not have

a restriction site in the gene. The sequences of the primers used for PCR are as follows:

Forward Primer- 5’-ACTGCGGATCCTCACCATGGACATCAGGCTC-3’

Bam HI

Reverse Primer- 5’-GCACTAAGCTTGTCATTTACCCGGAGAGTGG-3’

Hind III

3.20 REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION (RT-PCR)

3.20.1 Synthesis of cDNA from total RNA

Reagents

Tris-acetate EDTA buffer (TAE, 50X, 1 L)

Components Amount

Tris base 242 g

Glacial acetic acid 57.1 mL

EDTA 0.5 M

Water Upto 1000 mL


74

10X gel loading dye (10 mL)

Components Amount

Glycerol (50%) 5.0 mL

10% SDS 1.0 mL

Beta-mercaptoethanol 0.1 mL

Tris-Cl buffer (1.5 M) pH 6.8 2.0 mL

Bromophenol blue 0.04 g

Water 1.9 mL

Protocol

Reverse transcription of total RNA was performed in a total volume of 20 µl. To the 0.5 mL

DEPC treated microcentrifuge tube added the following in the order:

Reagents Amount

Sterile DEPC treated water 6 µl

RNAse inhibitor (RNasin 40 U/

µl)

1 µl

Total RNA 3 µl (~3 µg)

Reverse primer (10 µM) 2 µl

Heated to 70oC for 10 min. Quick chilled on ice and then added,

RNasin (40 U/µl) 1 µl

dNTP mix (10 mM) 2 µl

5X Reaction buffer 4 µl

RevertAid™ M-MuLV Reverse

transcriptase (200 U/µl)

1 µl

Mixed gently, incubated at 42oC for 1 hr. Terminated the reaction by

heating at 70oC for 15 min.

3.20.2 Polymerase chain reaction

Samples of cDNA were analysed by PCR amplification using specific nucleotide primers. The

reaction assay for 50 μl consisted of the following ingredients:


75

Reagents Amount

Nuclease free water 36.5 µl

cDNA from RT reaction(4 µg) 2.5 µl

Forward Primer (10 µM) 2.0 µl

Reverse Primer (10 µM) 2.0 µl

10X Reaction Buffer 5.0 µl

dNTP Mix (10 mM) 1.5 µl

Taq DNA polymerase (5 U/µl) 0.5 µl

Total volume 50 µl

The amplification was performed in a Master cycler® Thermocycler (Eppendorf, Germany)

using the following program

S. No. Program Cycles Temperature/Time

1. Denaturation 1 94˚C/2min

2. Denaturation

Annealing

Extension

40

94˚C/1min

58˚C/1min

72˚C /1min

3. Final extension 1 72˚C/10min

Initial denaturation of 94oC for 2 min followed by 40 cycles of denaturation at 94

oC for 1 min,

annealing at 58oC for 1 min and extension at 72

oC for 1 min. Final extension was carried out at

72oC for 10 min. The tubes were stored at -20˚C till further use. The PCR amplified product was

analyzed on agarose gel.

3.20.3 Agarose gel electrophoresis

0.8% gel was used for analysis of DNA. The appropriate amount of agarose was added to a flask

with 1X TAE buffer and boiled. The solution was cooled and ethidium bromide (0.5 μg/mL)

was added and the mixture was poured into a casting tray containing the sample comb and

allowed to solidify. After the gel had solidified, the comb was removed carefully and the gel was

mounted onto the electrophoresis chamber and covered with 1X TAE buffer. The PCR product

was mixed with 1X gel loading dye and loaded into the wells of the submerged gel. The gel was

run at a constant voltage of 80 V. The fragments were separated on the basis of molecular size


76

and conformation. After the gel was run ¾ of the length of the tray, it was visualized on UV

transilluminator.

3.20.4 Elution of the PCR amplified DNA fragment from agarose gel

The portion of the gel having the desired fragment was cut out and minced using a sterile scalpel

blade. The gel pieces were transferred to a microfuge tube and DNA was isolated from them

using nucleospin extract columns. Incubated 100 mg of agarose gel piece with 200 µl buffer NT

for 15 min at 50oC. The nucleospin extract II column was placed into a collection tube and

centrifuged for 1 min at 11,000xg. Flow through was discarded and 700 µl of wash buffer NT3

(containing ethanol) was added to the column. The column was again centrifuged for 1 min at

11,000xg. After complete removal of wash buffer, the spin columns were allowed to stand at RT

for 1-2 min. The nucleospin extract II column was placed into a new microcentrifuge tube, 20 µl

of buffer NE was added and incubated at RT for 1 min. The spin column was then centrifuged at

11,000xg for 1 min. The purified product was checked on 0.8% agarose gel and quantified.

3.21 CONSTRUCTION OF RECOMBINANT E. COLI EXPRESSION PLASMID

VECTOR

The 1.4kb gene was engineered to have Bam HI site at the 5` end and a Hind III site at the 3`

end to facilitate cloning into E. coli expression plasmid. The expression vector pRSETc (2.9kb)

has Bam H1 and Hind III sites at the multiple cloning site.

3.21.1 Restriction Enzyme Digestion of plasmid and PCR product with Bam HI:

To prepare vector and insert, the plasmid pRSETc and PCR product were first digested with

Bam HI.

(i) Restriction digestion was carried out by mixing the reagents as shown in the following:

Components pRSETc (Vector) PCR Product (Insert)

DNA 30 µl (20 µg) 100 µl

10X Reaction Buffer 20 µl 20 µl

RNase A (2 mg/mL) 5 µl -

Bam HI (10 U/µl) 3 µl 3 µl

Autoclaved water 142 µl 77 µl

Total 200 µl 200 µl


77

(ii) The reaction mixture was incubated at 37oC overnight.

(iii) To the sample 1/3 rd volume of 10 M ammonium acetate was added and incubated on ice

for 15 min to precipitate proteins.

(iv) After centrifugation at 10000rpm for 10 min, the DNA in the supernatant was precipitated

with equal volume of isopropanol and washed with 70% ethanol.

(v) The DNA pellet was dissolved in 177 µl of water and used for second digestion with Hind

III.

3.21.2 Restriction Enzyme Digestion of plasmid and PCR product with Hind III:

(i) To 177 µl of Bam HI digested DNA from (above step) 20 µl of 10X reaction buffer and 3 µl

of Hind III enzyme (10 U/µl) was added.

(ii) Incubated overnight at 37˚C.

(iii) To the sample 1/3 rd volume of 10 M ammonium acetate was added and incubated on ice

for 15 min to precipitate proteins. After centrifugation at 10000rpm for 10 min, the DNA in

the supernatant was precipitated with equal volume of isopropanol and washed with 70%

ethanol.

(iv) The PCR product (insert) was dissolved in 30 µl of TE buffer and pRSETc was dissolved in

26 µl of autoclaved DD water.

(v) This was followed by alkaline phosphatase treatment to remove the 5’ phosphate in the

vector.

3.21.3 Dephosphorylation of 5’ ends of the pRSETc vector

To 26 µl of DNA dissolved in autoclaved DD water, 3 µl of 10X Fast Alkaline phosphate buffer

and 1 µl of calf intestinal alkaline phosphatase enzyme was added. Sample was incubated at

37˚C for 20 min. The enzyme was inactivated by heating at 65-70oC for 15 min. Before ligation,

the digestion mixture was run on agarose gel and the required DNA fragment (Insert ~ 1.4 kb

and vector ~ 2.9 kb) was eluted from the gel and purified as described earlier (section 3.23.4).

3.21.4 Ligation of pRSETc vector with the insert DNA

Restriction enzyme digested vector and the insert DNA having compatible ends were added to a

20 μl reaction mixture as per the protocol described by Sambrook et al. (2001). A typical

ligation mixture contained:


78

Reagents Amount

10X DNA ligase buffer 1 μl (1X final concentration)

Vector DNA 100 ng (0.5 μl)

Insert DNA 3X molar excess (1 μl)

DNA ligase 0.5 μl (1-5 units)

Sterile water Upto 10 μl

The ligation reaction was carried out at 8˚C, overnight. After completion of the reaction, sample

was incubated in 65˚C water bath for 10 min to heat inactivate the enzyme.

3.21.5 Preparation of competent cells

Reagents

Luria Broth Medium (1 L, pH=7)

Components Amount

Tryptone 10 g

Yeast extract 5 g

Sodium chloride 10 g

Water 1000 mL

Sterilized media by autoclaving at 15 psi/121˚C for 20 min.

Luria Broth Agar (LA)*

*Added 1.5% agar to the LB medium and sterilized by autoclaving. The medium with agar

was allowed to cool down to 55˚C before antibiotics were added. Poured the plates directly

from the flask before the agar solidifies.

Calcium chloride (CaCl2) (0.1 M, 100 mL)

Components Amount

CaCl2 1.47 g

Water 100 mL

It was autoclaved for 15 min at 15 lbs / in2 at 121ºC, stored at 4C.


79

Protocol

Competent cells of E. coli were prepared by the method of Sambrook et al. (1989). All the steps

were carried out under sterile conditions. A single colony of E. coli DH5α was inoculated in 5

mL LB medium and grown overnight. The culture was diluted hundred times into 100 mL LB

medium and grown. When O.D at 600 nm (A600) reached 0.3-0.4, the culture was centrifuged at

4000rpm for 5 min at 4˚C. The supernatant was decanted and the pellet was resuspended gently

in 10 mL of ice-cold 100 mM CaCl2 solution. The sample was kept on ice for 30min and re-

centrifuged at 4000rpm for 5 min at 4˚C. Supernatant was decanted and the pellet was

resuspended gently in 2 mL of ice-cold 50 mM CaCl2 solution. The cells were stored overnight

at 4˚C prior to use, to increase the transformation efficiency.

3.21.6 Transformation of competent cells

Reagents

Ampicillin (100 mg/mL)

Components Amount

Ampicillin 100 mg


Sterilized by passing it through a 0.22 μM disposable filter. Dispensed the solution into

aliquots and stored them at -20˚C.

Protocol

Transformation of recombinant plasmid into competent E. coli DH5α cells was carried out

according to the protocol of Mandel and Higa (1970). All the steps were carried out under sterile

conditions. For transformation, 100 μl aliquots of competent cells were mixed with 10 μl DNA

samples (100-200 ng) and incubated on ice for 10 min. The cells were then subjected to heat

shock at 42˚C for 90 s and immediately placed in ice for 5 min. 1 mL of LB broth was added to

the cells and incubated at 37˚C with shaking for 1h. The cells were spinned at 6,000rpm for 1

min and the supernatant was spread on LB agar plates supplemented with ampicillin (100

μg/mL). The plates were incubated at 37˚C for 16-18 hr.


80

3.21.7 Isolation of plasmid DNA from E. coli DH5α

Reagents

Solution I (prepared fresh each time before use)

Components Amount

Tris-HCl, pH 8.0 25 mM

EDTA, pH 8.0 10 mM

Glucose 50 mM

Solution II (prepared fresh each time before use)

Components Amount

Sodium hydroxide (NaOH) 0.2 M

SDS 1%

Solution III

Components Amount

Potassium acetate, pH 4.8 3M

The pH of KAc solution was adjusted to 4.8 with glacial

acetic acid.

Protocol

Plasmid DNA was isolated by alkali lysis method as described by Bimboim and Dolly, (1979).

For screening of recombinant clones; individual colonies were inoculated in 2 mL LB broth

(mini scale isolation) containing 100 μg/mL ampicillin and cultures were grown at 37˚C

overnight at 200rpm in an incubator shaker. Cells were centrifuged at 8000rpm for 10 min at

4˚C and the supernatant was aspirated out. Pellet was resuspended in 200 μl of ice-cold Solution

I. It was allowed to stand on ice for 5 min. To the same tube, 400 μl of Solution II was added,

mixed gently by inverting twice or thrice and incubated at RT for 10 min. 300 μl of Solution III

was added to the tubes, mixed immediately and incubated in ice for 15 min. Sample was

centrifuged at 10,000rpm for 15 min at 4˚C. The supernatant was transferred to a fresh sterile

tube and 0.7 volume of isopropanol was added and was allowed to stand at RT for 2 h. The

DNA was recovered by centrifugation at 12,000rpm for 30 min at 4˚C. The DNA pellet was

washed with 70% ethanol and dissolved in 40 μl of sterile distilled water and stored at -20˚C for


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further use. To check for the presence of insert, these plasmids were subjected to double

digestion with BamHI and HindIII and looked for the release of 1.4kb fragment. The clone

which released 1.4kb fragment was selected.

3.22 EXPRESSION SCREENING OF CLONES HARBORING 55kDa RECOMBINANT

PROTEIN

Reagents

IPTG (1 M)

Components Amount

IPTG 2.38 g


Adjusted the volume of the solution to 10 mL with dH2O and

sterilized by passing it through 0.22 μM disposable filter.

Dispensed the solution into 1 mL aliquots and stored them at -

20˚C.

Protocol

The positive clones were selected and the minipreps of recombinant plasmid pRSETc isolated

from DH5α recombinant were used to transform E. coli expression host strain BL21 (DE3). The

BL21 (DE3) competent cells are an all purpose strain for high-level protein expression and easy

induction of T7 polymerase from lacUV5 promoter. Several of these clones were inoculated into

25 mL of LB broth containing 100 μg/mL ampicillin and grown at RT (25-30oC) overnight with

vigorous shaking. 200 mL of LB broth containing 100 μg/mL ampicillin was inoculated with 25

mL of overnight culture and was allowed to grow at RT with vigorous shaking for

approximately 4 hr (till OD600 is 0.6 to 0.8 is reached). At this stage, 1 mL of sample was

immediately taken out before induction (This sample is used as an un-induced control).The

expression of recombinant protein was induced by adding IPTG to the rest of the culture media

at a final concentration of 1 mM and incubating the culture for another 4 hr at RT with shaking.1

mL sample was again taken out before proceeding to the next step (This sample is used as

induced sample).The cells from both the uninduced and induced samples were harvested by

centrifugation at 7,000rpm for 5 min. The cell pellet was stored at -20oC until further use. After

induction, equivalent number of cells from different cultures (normalized on the basis of A 600


82

values) was lysed in 100 µl of 2X SDS sample buffer. The samples were boiled in water bath for

5 min and centrifuge at 10000rpm at RT for 5 min and analyzed by SDS-PAGE. Uninduced

cultures were analyzed in parallel. One clone that expressed maximum levels of the recombinant

protein was chosen for further study.

3.23 PURIFICATION OF 55kDa RECOMBINANT PROTEIN BY Ni-NTA AFFINITY

CHROMATOGRAPHY

Principle

Ni-NTA superflow columns contain a Ni-NTA silica matrix for purifying recombinant proteins

carrying a 6x His Tag from cell lysates using gravity flow or a vacuum driven automated

procedure. Histidine residues in the 6x His Tag bind to the vacant positions in the co-ordination

sphere of the immobilized nickel ions with high specificity and affinity. High levels of

expression of recombinant proteins in a variety of expression systems can lead to the formation

of insoluble aggregates in E. coli, these are known as inclusion bodies. Such inclusion bodies

can be lysed under denaturing conditions to obtain the purified protein. Figure 3.1 outlines the

general strategy that may be followed for isolation of the induced protein.

Figure 3.1 Strategy for purifying 6x his tag on Ni-NTA column


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Reagents

8X Charge buffer- (400 mM NiSO4), dilute 1 in 8 before use.

Binding Buffer (1X, 100 mL)

Components Amount

NaCl 500 mM

Tris-HCl (pH 7.9) 20 mM

Imidazole 5 mM

Urea 6 M

For preparing 100 mL of 1X binding buffer, mixed all the components

(NaCl, Tris-HCl, Imidazole) in 40 mL DD water and added 6 M Urea at the

end and dissolved completely. Made up the volume to 100 mL using DD

water.

Wash Buffer (1X)

NaCl Tris-HCl (pH-7.9) Imidazole Urea

Wash Buffer I 500 mM 20 mM 80 mM 6 M

Wash Buffer II 500 mM 20 mM 120 mM 6 M

Wash Buffer III 500 mM 20 mM 180 mM 6 M

Elution Buffer (1X)

Components Amount

NaCl 500 mM

Tris-HCl (pH 7.9) 20 mM

Imidazole 500 mM

Urea 6 M

Lysis/Homogenisation Buffer

Components Amount

Sodium phosphate buffer (pH- 8.0) 10 mM

SDS 0.1%

Urea 6 M


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Protocol

Purification of the induced protein under denaturing conditions using Ni-NTA affinity

chromatography was essentially done as described by Khanam et al. (2006) with minor

modifications. The induced culture cell pellet (approximately 1.5 g wet weight) was lysed by re-

suspending in 10 mL ice-cold lysis/homogenisation buffer (10 mL buffer for 250 mL culture

worth cells), vortexed and homogenized 6-8 times (Polytron 3100 homogenizer). The lysate was

then centrifuged at 10,000rpm for 45 min at 4oC. The resulting supernatant was transferred to

fresh, autoclaved bottle and used for Ni-NTA affinity chromatography.

Ni-NTA column was prepared by washing with distilled water and methanol and charging it

with 5 mL of 1X charge buffer containing NiSO4. Column was washed with 1X binding buffer

(15 mL). After wash with 1X binding buffer, protein was loaded in aliquots of 1 mL at a time.

This suspension was gently rocked overnight at 4˚C for 2-3 hrs and then packed into a column.

6x His tagged proteins are bound while other proteins pass through the matrix. After collecting

the flow through, the column was washed extensively with 20 volumes of 1X wash buffer I

(having concentration of imidazole-80 mM) and was collected. Again column was washed with

10 volumes of 1X wash buffer II (having imidazole-120 mM) and was collected. Again column

was washed with 5 volumes of 1X wash buffer III (having concentration of imidazole-180 mM)

and was collected. Elution was done using 15 volumes of 1X Elute buffer (having concentration

of imidazole-500 mM) and was collected. The eluted samples were stored at -80C and 10%

SDS PAGE was run and western blot analysis was done thereafter.

3.24 VALIDATION OF RECOMBINANT PROTEIN IMMUNOGLOBULIN GAMMA1

HEAVY CHAIN (IgG1H) BY IMMUNOBLOTTING

3.24.1 Immunoblot analysis of the His-tagged recombinant protein (55kDa) with anti His-

tag antibody

To validate that the expressed protein is his-tagged recombinant protein (Immunoglobulin

gamma1 heavy chain; 55kDa) western blotting was carried out. Eluted and purified samples of

recombinant protein (25 μg) were run on 10% SDS-PAGE and transferred on nitrocellulose

membrane at 100 V, 4°C for 2-3 hr. After the transfer, the blot was blocked with blocking

solution containing 3% BSA (Bovine Serum Albumin) and 0.1% Tween-20 in Tris-buffered

saline (TBS) for 1 hr at RT. The membrane was then incubated with primary monoclonal anti


85

His Tag antibody (final dilution 1:1000) in blocking solution at 4°C for 1 hr, with shaking. The

membrane was rinsed thrice with 0.1% Tween-20 in TBS and incubated with Goat anti mouse

IgG-HRP conjugated secondary antibody (final dilution 1:1000) and kept for mixing at RT for 1

hr. The membrane was again washed with 1X TBS thrice and developed by adding the substrate

DAB and H2O2.

3.24.2 Immuno Blot analysis and validation of antibody response against the recombinant

protein Immunoglobulin gamma1 heavy chain in rat sera

To validate the presence of antibodies against the recombinant protein immunoglobulin gamma1

heavy chain in rat sera, western blot analysis was carried out according to the protocol in section

3.24.1 with minor modifications. Purified samples of recombinant protein (25 μg) were run on

10% SDS-PAGE and transferred on nitrocellulose membrane at 100V, 4°C for 2-3 hr. After the

transfer, the blot was blocked with blocking solution containing 3% BSA (Bovine Serum

Albumin) and 0.1% Tween-20 in Tris-buffered saline (TBS) for 1 hr at RT. The membrane was

then incubated with rat sera at 1:50 dilution (from NNK treated groups; 1 month, 3 months, 6

months and 9 months and control) as primary antibody in blocking solution at 4°C for 1 hr, with

shaking. The membrane was rinsed thrice with 0.1% Tween-20 in TBS and incubated with

rabbit anti-rat IgG1-HRP conjugated secondary antibody (final dilution 1:1000) and kept for

mixing at RT for 1 hr. The membrane was again washed with 1X TBS thrice and developed by

adding the substrate DAB and H2O2.

3.1 reagents and chemicals name sourceshodhganga.inflibnet.ac.in/bitstream/10603/11720/1/10....

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