UNIVERSITI TEKNOLOGI MARA FAKULTI KEJURUTERAAN KIMIA

CHEMICAL ENGINEERING LABORATORY (CHE465)

No. Title Allocated Marks (%) Marks

1 Abstract/Summary 5  2 Introduction 5  3 Aims 5  4 Theory 5  5 Apparatus 5  6 Methodology/Procedure 10  7 Results 10  8 Calculations 10  9 Discussion 20  10 Conclusion 5  11 Recommendations 5  12 Reference / Appendix 5  13 Supervisor’s grading 10  

TOTAL MARKS 100  Remarks:

Checked by : Rechecked by:

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Date : Date :

1

NAME : MARISSA DE VALDA BT MOHD YATIM

STUDENT NO : 2013229382

GROUP : GROUP 1

EXPERIMENT : ESTIMATION OF PROTEIN

DATE PERFORMED : 15 OCTOBER 2014

SEMESTER : 3

Table of Content

Content Page

1 Abstract/Summary 3

2 Introduction 4-5

3 Aims 5

4 Theory 6-7

5 Apparatus 8

6 Procedure/Methodology 8-9

7 Results and Discussions 10-17

8 Calculation 17

9 Conclusion and Recommendation 18-19

10 References 20

11 Appendix 21-22

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ABSTRACT

In chemistry, acids and bases have been defined differently by three sets of theories. One

is the Arrhenius definition, which revolves around the idea that acids are substances that ionize

(break off) in an aqueous solution to produce hydrogen (H+) ions while bases produce hydroxide

(OH-) ions in solution. On the other hand, the Bronsted-Lowry definition defines acids as

substances that donate protons (H+) whereas bases are substances that accept protons. Also, the

Lewis theory of acids and bases states that acids are electron pair acceptors while bases are

electron pair donors. Acids and bases can be defined by their physical and chemical observations

[1].

The objectives of this experiment are to study the method of assay preparation in protein

estimation and to determine the best assay in protein estimation.

There are four methodology employed in determining protein concentrations which are

spectrophotometric assay, Biuret, Lowry and Bradford assays. We could complete all assays but

the biuret as we are short of the chemicals that are going to be used for that assay.

At the end of the experiment, it was observed that in Lowry assay, a blue colored product

is formed by using reagent Folin-Ciocalteu reagent that is used in addition to strengthen the

colour. In Bradford’s assay, blue color is also formed by using Coomassie Blue reagent that is

used to bind to proteins in acidic solution.

In conclusion, the most sensitive technique is the Bradford method. It is highly sensitive,

is able to measure 1-20 µg of protein and is very fast. Only relatively few materials interfere with

it (it works even in presence of urea or guanidine hydrochloride) but, importantly, detergents do.

Even traces of detergent (e.g. cleaning products) can invalidate the results. Its disadvantages are

that it depends strongly on amino acid composition and that it stains the cuvettes used.Lowry

assay. This method is quite sensitive and is able to detect even 1 µg of protein [3].

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1. INTRODUCTION

Protein assays, most notably quantitation or estimation assays, for determining protein

concentration are one of the most widely used methods in life science research. Protein

estimation of protein concentration is necessary in protein purification, electrophoresis, cell

biology, molecular biology, and other research applications. Although there are a wide variety of

protein assays available, none of the assays can be used without first considering their suitability

for the application. Each method has its own advantages and limitations and often it is necessary

to obtain more than one type of protein assay for research applications. The assays used in this

experiment are Bradford, Lowry and Spectrophotometric Assay.

The Bradford protein assay is one of several simple methods commonly used to

determine the total protein concentration of a sample. The method is based on the proportional

binding of the dye Coomassie to proteins. Within the linear range of the assay (~5-25 mcg/mL),

the more protein present, the more Coomassie binds. Furthermore, the assay is colorimetric; as

the protein concentration increases, the color of the test sample becomes darker. Coomassie

absorbs at 595 nm. The protein concentration of a test sample is determined by comparison to

that of a series of protein standards known to reproducibly exhibit a linear absorbance profile in

this assay. Although different protein standards can be used, we have chosen the most widely

used protein as our standard - Bovine Serum Albumin (BSA) [2].

Lowry’s assay for total protein is one of the most commonly performed colorimetric

assays. This procedure is sensitive because it employs two colour forming reactions. It involves

reactions in which Cu2+ in presence of a base reacts with a peptide bond of protein under

alkaline conditions resulting in reduction of cupric ions (Cu2+) to cuprous ions (Cu+), and

Lowry’s reaction in which the Folin Ciocaltaeu reagent which contains phosphomolybdic

complex which is a mixture of sodium tungstate, sodium molybdate and phosphate, along with

copper sulphate solution and the protein, a blue purple colour is produced which can be assessed

by measuring the absorbance at 650-700nm.

Spectrophotometric assay is based on the fact that two of the aromatic amino acids,

tryptophan and tyrosine, show a peak in absorbance around 280 nm. It has the advantage of

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being quick and easy. Since it needs no chemical reaction to be performed, it is widely used for

detection of proteins or peptides during their separation by chromatography. As proteins contain

different ratios of aromatic amino acids, per se it is more suited to the comparison of solutions of

the same protein and less to absolute measurement. The latter requires the knowledge of the

molar extinction coefficients of proteins. For many proteins, these were determined and can be

found in the literature. Moreover, if we know the number of tyrosine and tryptophan amino acids

in the protein of interest, since their absorption values are additive, it is possible to calculate the

molar extinction coefficient.

2. OBJECTIVES

The objectives of this experiment are:

i. To study the method of assay preparation in protein estimation.

ii. To determine the best assay in protein estimation.

iii. To determine protein concentrations.

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3. THEORY

The Lowry protein assay is named after Oliver H. Lowry, who developed and introduced it

(Lowry, et al., 1951). It offered a significant improvement over previous protein assays and his

paper became one of the most cited references in life science literature for many years. The

Modified Lowry Protein Assay uses a stable reagent that replaces two unstable reagents

described by Lowry. Essentially, the assay is an enhanced biuret assay involving copper

chelation chemistry [4].

Use of Coomassie G-250 dye in a colorimetric reagent for the detection and quantitation of

total protein was first described by Dr. Marion Bradford in 1976 (Bradford, 1976). Thermo

Scientific Pierce Coomassie and Coomassie Plus Protein Assay Products are variants of the

reagent first reported by Bradford

Figure 1: Chemical structure of Coomassie dye

In the acidic environment of the reagent, protein binds to the Coomassie dye. This results

in a spectral shift from the reddish/brown form of the dye (absorbance maximum at 465nm) to

the blue form of the dye (absorbance maximum at 610nm). The difference between the two

forms of the dye is greatest at 595nm, so that is the optimal wavelength to measure the blue color

from the Coomassie dye-protein complex. If desired, the blue color can be measured at any

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wavelength between 575nm and 615nm. At the two extremes (575nm and 615nm) there is a loss

of about 10% in the measured amount of color (absorbance) compared to that obtained at 595nm

[4].

Protein concentration can also be determined from the protein’s own (intrinsic) UV

absorbance. Note, however, that these methods may give different results for different proteins of

the same concentration. Also, different methods can yield somewhat different results for the

same protein. There is no absolute photometric protein concentration assay. All methods have

advantages and disadvantages and we must choose among them by taking the following aspects

into consideration: specificity, sensitivity, the measurable range of concentration, the accuracy,

the nature of the protein to be examined, the presence of materials interfering with the

measurement, and the time required for the measurement.

Figure 2: Example of a Spectrophotometer used in laboratories

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4. METHODOLOGY

4.1 Materials and Apparatus

The materials and apparatus used in this experiment are:

- Biuret reagent- Lowry reagent- Bradford reagent- Protein- 2% sodium carbonate- 0.4% NaOH- Folin-Ciocalteu phenol (folin) reagent- Distilled water

- Spectrophotometer- Pipette- Beaker- Cuvettes

4.2 Procedures

a) Preparation of reagents

1. All series were included a zero protein (water) tube (reagent blank).

2. Lowry: 0.25 mL of protein was mixed with 2.5 mL of Lowry’s reagent. After 10

minutes, 0.25 mL of Lowry reagent 2 was added and mixed well immediately.

After 30 minutes, the absorbance was measured at 750 nm.

3. Bradford: 0.25 mL of protein was mixed with 2.5 mL of Bradford’s reagent and

the absorbance was measured at 595 nm.

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b) Lowry Reagents

Reagent 1:

One volume of reagent B (0.5% copper sulfate pentahydrate, 1% sodium or potassium tartrate) was mixed with 50 volumes of reagent A (2% sodium carbonate, 0.4% NaOH).

Reagent 2:

Commercial Folin-Ciocalteu phenol reagent was diluted with an equal volume of water.

To quantify protein: 0.25 mL of protein was mixed with 2.5 mL of Lowry’s reagent. After 10 minutes, 0.25 mL of Lowry reagent 2 was added and mixed well immediately. After 30 minutes, the absorbance was measured at 750 nm.

c) Bradford's Reagent

100 mg Coomassie Blue G-250 was dissolved in 50 mL of 95% ethanol, and 100 mL of 85% phosphoric acid was added before being diluted to one liter. The reagent was filtered once as it seems to precipitate dye over time.

To quantify protein: 0.25 mL of protein was mixed with 2.5 mL of Bradford’s

reagent and the absorbance was measured at 595 nm after 5 minutes.

Disadvantages: A high blank which may affect subsequent readings because some reagent adheres to the cuvette.  Another is that it is very sensitive to the presence of detergent e.g. from poorly-rinsed glassware.

d) Data Analysis

Separate graphs for each assays were plotted based on the data obtained.

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5. RESULTS

A. Lowry’s Reagent Method

i. Bovine Serum Albumin ( BSA )

Concentration(mg/ml)

Absorbance

0.00 0.000

0.12 0.401

0.16 0.504

0.20 0.507

0.24 0.676

0.28 0.585

0.30 0.511

10

0 0.05 0.1 0.15 0.2 0.25 0.3 0.350

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

R² = 0.761884638363958

Absorbance vs Concentration of BSA

Concentration of BSA (mg/ml

Abso

rban

ce

ii. Gelatin

Concentration(mg/ml)

Absorbance

0.0 0.000

0.2 0.097

0.4 0.227

0.6 0.235

0.8 0.368

1.0 0.506

11

0 0.2 0.4 0.6 0.8 1 1.20

0.1

0.2

0.3

0.4

0.5

0.6

R² = 0.970021580124689

Absorbance vs Concentration of Gelatin

Concentration of Gelatin (mg/ml)

Abso

rban

ce

The strong blue colour is created by two reactions that is first, the formation of the

coordination bond between peptide bond nitrogens and a copper ion and secondly, the reduction

of the Folin-Ciocalteu reagent by tyrosine (phosphomolybdic and phosphotungstic acid of the

reagent react with phenol). The measurement is carried out at 750 nm. A calibration curve is

created and the concentration of the unknown protein is determined from the curve [2].

B. Bradford’s Reagent Method

i. Bovine Serum Albumin ( BSA )

Concentration(mg/ml)

Absorbance

0.00 0

0.12 0.412

0.16 0.649

0.20 0.773

0.24 0.994

0.28 1.120

12

0.30 1.136

0 0.05 0.1 0.15 0.2 0.25 0.3 0.350

0.2

0.4

0.6

0.8

1

1.2R² = 0.991522377713978

Absorbance vs Concentration of BSA

Concentration of BSA (mg/ml

Axis

Title

ii. Gelatin

Concentration(mg/ml)

Absorbance

0.0 0.000

0.2 0.289

0.4 0.547

0.6 0.879

0.8 0.936

1.0 1.120

13

0 0.2 0.4 0.6 0.8 1 1.20

0.2

0.4

0.6

0.8

1

1.2R² = 0.966907449269794

Absorbance vs Concentration of Gelatin

Concentration of Gelatin (mg/ml

Axis

Title

Bradford assay, a colorimetric protein assay, is based on an absorbance shift of the dye

Coomassie Brilliant Blue G-250 in which under acidic conditions the red form of the dye is

converted into its bluer form to bind to the protein being assayed. During the formation of this

complex, two types of bond interaction take place: the red form of Coomassie dye first donates

its free electron to the ionizable groups on the protein, which causes a disruption of the protein's

native state, consequently exposing its hydrophobic pockets. These pockets in the protein's

tertiary structure bind non-covalently to the non-polar region of the dye via van der Waals forces,

positioning the positive amine groups in proximity with the negative charge of the dye. The bond

is further strengthened by the ionic interaction between the two. The binding of the protein

stabilizes the blue form of the Coomassie dye; thus the amount of the complex present in

solution is a measure for the protein concentration, and can be estimated by use of an absorbance

reading [5].

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C. Spectophotometry Assay

i. Bovine Serum Albumin ( BSA )

Concentration(mg/ml)

Absorbance

0.00 0.000

0.12 0.019

0.16 0.142

0.20 0.177

0.24 0.242

0.28 0.327

15

0.30 0.359

0 0.05 0.1 0.15 0.2 0.25 0.3 0.350

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

R² = 0.901632561377876

Absorbance vs Concentration of BSA

Concentration of BSA (mg/ml)

Abso

rban

ce

ii. Gelatin

Concentration(mg/ml)

Absorbance

0.0 0.000

0.2 0.004

0.4 0.035

0.6 0.044

0.8 0.078

1.0 0.081

16

0 0.2 0.4 0.6 0.8 1 1.20

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

R² = 0.953340456288103

Absorbance vs Concentration of Gelatin

Concentration of Gelatin (mg/ml)

Abso

rban

ce

This method is based on the fact that two of the aromatic amino acids, tryptophan and

tyrosine, show a peak in absorbance around 280 nm. It has the advantage of being quick and

easy. Since it needs no chemical reaction to be performed, it is widely used for detection of

proteins or peptides during their separation by chromatography. As proteins contain different

ratios of aromatic amino acids, per se it is more suited to the comparison of solutions of the same

protein and less to absolute measurement. The latter requires the knowledge of the molar

extinction coefficients of proteins. For many proteins, these were determined and can be found in

the literature. Moreover, if we know the number of tyrosine and tryptophan amino acids in the

protein of interest, since their absorption values are additive, it is possible to calculate the molar

extinction coefficient [2].

6. CALCULATION

a. Bovine Serum Albumin (BSA)

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Concentration

(mg/ml)

Volume of BSA

(ml)

Volume of Water

(ml)

0.00 0 250

0.12 30 220

0.16 40 210

0.20 50 200

0.24 60 190

0.28 70 180

0.32 80 170

b. Gelatin

Concentration

(mg/ml)

Volume of Gelatin

(ml)

Volume of Water

(ml)

0.00 0 100

0.20 20 80

0.40 40 60

0.60 60 40

0.80 80 20

1.00 100 0

7. CONCLUSION AND RECOMMENDATION

From the findings of this experiment, it can be concluded that most commercial protein

assay reagents are well-characterized, robust products that provide consistent, reliable results.

Nevertheless, each assay reagent has its limitations; having a basic understanding of the

chemistries involved with each type of assay is essential for selecting an appropriate method for

a given sample and for correctly evaluating results.

Bradford assay

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Bradford assays are the fastest and easiest to perform of all protein assays. The assay is

performed at room temperature and no special equipment is required. Resultant blue color is

measured at 595nm following a short room temperature incubation. The Coomassie dye-

containing protein assays are compatible with most salts, solvents, buffers, thiols, reducing

substances and metal chelating agents encountered in protein samples.

The main disadvantage of Coomassie based protein assays is their incompatibility with

surfactants at concentrations routinely used to solubilize membrane proteins. In general, the

presence of a surfactant in the sample, even at low concentrations, causes precipitation of the

reagent. In addition, the Coomassie dye reagent is highly acidic, so proteins with poor acid-

solubility cannot be assayed with this reagent. Finally, Coomassie reagents result in about twice

as much protein-to-protein variation as copper chelation-based assay reagents.

The recommendation is that the ready-to-use liquid Coomassie dye reagents should be

mixed gently by inversion just before use. The dye in these liquid reagents forms loose

aggregates within 60 minutes in undisturbed solutions. Gentle mixing of the reagent by inversion

of the bottle will uniformly disperse the dye and ensure that aliquots are homogeneous.

Lowry Assay

The assay is performed in two distinct steps. First, protein is reacted with alkaline cupric

sulfate in the presence of tartrate for 10 minutes at room temperature. During this incubation, a

tetradentate copper complex forms from four peptide bonds and one atom of copper (this is the

"biuret reaction"). Second, a phosphomolybdic-phosphotungstic acid solution is added. This

compound (called Folin-phenol reagent) becomes reduced, producing an intense blue color.

The final blue color is optimally measured at 750nm, but it can be measured at any

wavelength between 650nm and 750nm with little loss of color intensity. It is best to measure the

color at 750nm since few other substances absorb light at that wavelength.

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The recommendation is that the Folin phenol reagent must 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. The Modified Lowry

Protein Assay Reagent must be refrigerated for long-term storage, but it must be warmed to room

temperature before use. Using cold Modified Lowry Protein Assay Reagent will result in low

absorbance values.

Spectrophotometric Assay

This method is based on the fact that two of the aromatic amino acids, tryptophan and

tyrosine, show a peak in absorbance around 280 nm. It has the advantage of being quick and

easy. Since it needs no chemical reaction to be performed, it is widely used for detection of

proteins or peptides during their separation by chromatography. As proteins contain different

ratios of aromatic amino acids, per se it is more suited to the comparison of solutions of the same

protein and less to absolute measurement. The latter requires the knowledge of the molar

extinction coefficients of proteins. For many proteins, these were determined and can be found in

the literature. Moreover, if we know the number of tyrosine and tryptophan amino acids in the

protein of interest, since their absorption values are additive, it is possible to calculate the molar

extinction coefficient.

REFERENCES

[1] Petrucci, Ralph H. General Chemistry: Principles and Modern Applications. Macmillian:

2007.

[2] Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein

utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976.

[3] Berg, J.M., Tymoczko, J.L., Stryer, L.: Biochemistry (2012) 7th edition, W. H. Freeman and

Company, New York; ISBN-13: 9781429229364

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[4] Krohn, R.I. (2002). The Colorimetric Detection and Quantitation of Total Protein, Current

Protocols in Cell Biology , A.3H.1-A.3H.28, John Wiley & Sons, Inc.

[5] Dennison, Clive (2003), A guide to protein isolation, Focus on structural biology 3: 39, ISBN

1402012241

APPENDIX

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Figure 3: Bradford’s reagent Figure 4: Lowry’s reagent

Figure 5: Spectrophotometric assay Figure 6: Bradford assay

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Figure 7: Lowry assay

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