The Bradford Method for Determining Protein Concentrations

Introduction
Aim:
The aim of this project is to determine the concentration of the unknown protein solutions of the standard curve, by using the Bradford method.

Theory and Principles:

Principles The Bradford method uses a dye, Coomassie Brilliant Blue, to determine the protein concentration. It has a high hydrophilic nature, as it has various hydrocarbons and carbon rings that stabilises its ionic form and causes a visible colour change. It also has an anionic nature because of the presence of the sulphuric acid groups. The assay is based on the principle that the absorbance maximum of an acidic solution of Coomassie Brilliant Blue will shift to a higher value (from 465nm to 595nm) when it binds to a protein. The absorbance at 595nm is a direct relation to the concentration of the protein in the solution.

Figure 1: Structure of Coomassie Brilliant Blue G-250

The positively charged (basic) amino acids will react with the dye. Coomassie Brilliant Blue has been known to primarily react with Arginine and to a lesser extent, with Lysine and Histodine. There is only a limited reaction with the aromatic acids Tyrosine, Tryptophan and Phenylalanine. The basic amino acids will carry a large positive charge in an acidic pH solution, because of the strong electrostatic interaction between the amino acids and the SO3groups of the dye. To compare different proteins however, it must be assumed the determinants that are necessary for the reaction are all equally presented by all the different proteins. Therefore we must use the methods for protein determination to employ surface

change manipulation and hydrophilic character of the solvent to convert the protein structure to random coils. It is also assumed that all proteins will take to the random coil structure to the same degree.

Experiment The Bradford Method

Materials and Reagents:
100 mg Coomassie Brilliant Blue G250 100ml of 85% phosphoric acid 50ml of 95% ethanol 50ml 1M NaOH. Distilled Water Either BSA or BIg standard solutions Unknown Solution Working Bradford reagent (10ml of stock Bradford reagent diluted with 250ml distilled water)

Apparatus
Visible light spectrophotometer Disposable cuvettes (although the dye will stick to these surfaces. It might be advisable to use plastic and glassware instead) 5 test tubes Volumetric flasks

Procedure
Reagents:
1. Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol; add 100 ml 85% (w/v) phosphoric acid. Dilute to 1 litre when the dye has completely dissolved, and filter through Whatman #1 paper just before use. 2. (Optional) 1 M NaOH (to be used if samples are not readily soluble in the colour reagent). The Bradford reagent should be a light brown in colour. Filtration may have to be repeated to rid the reagent of blue components. After filtration, store the reagent in an amber bottle and keep at room temperature. Making Protein stock solutions:

1. Stock Bovine Serum Albumin (BSA) solution (10mg/ml): Dilute 0.2g BSA in 20ml distilled water. 2. Stock Bovine Immunoglobulin (Blg) solution (10mg/ml): Dilute 0.2g BSA in 20ml distilled water. The working BSA and Blg range is: 0.5 1.25mg/ml Plastic and glassware used during this assay should be clean and free of detergents. 1. Prepare the following test tubes.

(For this project, it is decided that BSA will be used)

Table 1: Preparation of test tubes for Bradford method Distilled Water 0.25 mg/ml Protein 0.5 mg/ml Protien 0.75 mg/ml Protien 1.0 mg/ml Protien 1.25 mg/ml Protien Unknown Working Bradford 2. 3. Blank 0.1 ml Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Unknown

0.1 ml

0.1 ml 0.1 ml

0.1 ml 0.1 ml

4ml

4ml

4ml

4ml

4ml

4ml

0.1 ml 4ml

Thoroughly mix after every addition of Bradford regent. Incubate the solution for 5min at room temperature. Throw the solution over to a small test tube. Fit the test tube inside the Visible light spectrophotometer. (Remember to calibrate the Visible light spectrophotometer with distilled water, before using it to determine the values of the other solutions) Make sure the solution is high enough to allow the light to pass through. (Inadequate amounts of solution will result in a faulty reading) Read the absorbance of the blank and test solutions at 595nm. Make notes of the values and draw a standard graph.

Data and Calculations:

Table 2: Absorbance readings for different proteins at 595nm, using the Bradford method: Protein Concentration (mM) x-axis 0.00 0.10 0.20 0.40 0.80 Unknown Absorbency (595nm) y-axis 0.00 0.15 0.25 0.50 0.95 0.35

Graph 1: Protein Standard Curve

Protein Standard curve

1.20 1.00 Absorbancy (595nm)

0.95

0.80

0.60 0.50 0.40 0.25 0.15 0.00 0.00 0.20 0.40 0.60 0.80 1.00 Protein Concentration(mM)

0.20

0.00

Determining the Unknown:

(Reading estimate values from the graph) Incline (m) = y2 - y1 / x2 x1
= 0.5 0.23 / 0.4 0.2

= 0.27 / 0.2 m = 1.35 (Determining c) 0.23 = 1.35(0.35) + c C = 0.0621 (Incorporating the Unknown) 0.35 = 1.35 (x) + 0.0621 0.2592593 = x + 0.0621 (x = 0.1971593) x = 0.19mM unknown protein

Results and Discussion:

Table 3: Final readings for the different concentrations of proteins:
(Protein) (mg/ml) Abs. BSA 0.00 0.00 0.1 0.15 0.2 0.25 0.4 0.50 0.8 0.95 0.19 0.35

About Amino acids: Amino acids are the building blocks that make up proteins. They are linked together with peptide bonds to form the primary structure of the protein. Mechanisms lead to the production of secondary, tertiary and quaternary structures of the protein. The Native proteins are large. Mechanisms are determined by the amino acid sequence of the protein. Amino acids determine the shape of the protein. They also have a direct effect on the function of that protein. All amino acids have a -carbon to which hydrogen, amino, carboxyl or an R-group is attached.

There are 20 different amino acids. The difference in properties of the R-groups has been used to divide the amino acids into 5 different groups. Firstly, the nonpolar group; aliphatic amino acids with hydrocarbon hydrophobic R-groups. The second group is the aromatic amino acids with aromatic ring structured R-groups. The third group has polar, but uncharged amino acids, with R-groups containing oxygen, sulphur or nitrogen polar groups. Finally, the fourth and fifth groups also contain oxygen, sulphur and nitrogen atoms but are polar in nature. These groups also have a pH value, which is positive for the amino acids in group 4, but negative for those in group 5.

The amino acid pH of the fourth and fifth groups are also dependant on the surrounding pH. Here the pI of the amino acid is relevant. The more the pI exceeds the surrounding pH, the more positive such an amino acid population becomes. If the surrounding pH is much higher than the pI of the amino acid, the population will become negative. Negative amino acids have pI values that are in the low pH range. They have donated most of their hydrogen groups to the surrounding environment and is said to be acidic. On the other hand, positively charged amino acids have pI values in the basic pH range, and have a restricted hydrogen contribution to the surrounding solution. Furthermore, it is important to note, that the amino acids will show a difference in chemical reactivity, because of the difference in their R-groups. They will thus react differently towards the same reactant. The Bradford method: There are various ways of determining the concentration of proteins, for example the Bradford method, the Biuret method and the Lowry method. Although all of these are fairly accurate, the Bradford method can be retested within minutes and also uses less of the protein than Lowry. Normally the Coomassie Brilliant Blue G-250 dye reagent reacts primarily with arginine, and less with histidine, tyrosine, tryptophan and phenylalanine. The assay is also less accurate for basic and acidic proteins. The Bradford Method is more sensitive to Bovine Serum Albumin (BSA) than other proteins, by about a factor of two. That is why it was used in this specific project. The addition of NaOH was suggested by Stotscheck (1990) to allow the stabilisation of membrane proteins and to reduce the protein-to-protein variation in colour yield. By using the Bradford Method, quick and relatively easy tests can be done to determine and compare the concentrations of different proteins. It is even possible to determine the exact protein used, if the correct information is at hand. The Bradford uses less reagents and proteins than other methods, and is therefore cost and time effective.

The information gained by testing the various concentrations of BSA could be used to set up a graph. Calculations, using the graph, could be made, and we were able to approve the aim, by finding the concentration of the unknown protein. Various tests were done, so that the average could be determined and used, rather than trust one test alone. A blank test was also done, to set the standard for the other tests. Comparisons could be made and greater precision achieved.

Conclusion:
By using the Bradford Method, it is able to determine the concentration of an unknown protein. Data collected from the tests, can be tabulated and graphed, to make calculating the concentration easier. The Bradford Method is a cost and time effective method that gives reliable results. It is also easy to determine the desired information, as a linear graph can be drawn from the test results.

References:
1. BCM 254/256 Practical Guide, Department of Biochemistry, Faculty of Natural and Agricultural Sciences, University of Pretoria 2011

2. http://bioteachnology.com/protein/bradford-method-colorimetric-proteinassay [Accessed site 1 March 2011]

3. Bradford, MM. A rapid and sensitive for the quantisation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976.

4. http://www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html [Accessed site 1 March 2011]