The 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. The (bound) form of the dye has an absorption spectrum maximum historically held to be at 595 nm. Objective: To determine the technique to a series of protein purification steps that yield ?-lactalbumin in sufficient amounts and purity for futher characterization by UV spectroscopy.
Introduction: Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification.
The most abundant protein found in milk is caseins and it can be removed by heat and low pH. The most general method to monitor the purification process is by running a SDS-polyacrylamide gel electrophoresis of the different steps. This method only gives a rough measure of the amounts of different proteins in the mixture, and it is not able to distinguish between proteins with similar apparent molecular weight. the purest, isolated form of bovine alpha-lactalbumin commercially available. Alpha-lactalbumin is the primary protein in human milk, and is therefore extremely important for infant nutrition.
The structure of alpha-lactalbumin is well known and is composed of 123 amino acids and 4 disulfide bridges. The molecular weight of alpha-lactalbumin is 14. 2K Daltons. Alpha lactalbumin regulates lactose biosynthesis by forming the lactose synthase complex with beta 1,4 galactosyltransferase. Method: Session 1 1. About 100ml of fresh milk was added into a 1liter beaker and heated to 40°C in a water bath with gentle stirring. 2. Then, 20g of anhydrous sodium sulphate were added slowly, a little at a time. The addition of sodium sulphate was carried out over 10 to 15 minutes.
After the addition, it was incubated for a further 10minutes. . After that, the solution was filtered using a Buchner funnel layered with cheese cloth (3-4 layers). Filtered under suction and kept the filtrate. (1ml of the filtrate was kept in an eppendorf tube and stored at -20°C for further analysis. Labelled it as S1). 4. The concentrated HCL was added drop wise to the remaining filtrate in a 1liter beaker until the pH reaches 3. 0 using pH meter with gentle stirring. 5. Later, the mixture was centrifuged at 10,000 x g for 15 minutes and kept the precipitate. The precipitated was stored at -20°C until the next laboratory session. The tube was labelled before storing in the freezer.
Session 2 A) Purification procedure (continued) 1. The precipitate was thawed out slowly by running it under tap water. Then, 10ml of distilled water was added to dissolve the precipitate. (1ml of the mixture was kept in an eppendorf tube as before and labelled it S2). 2. To the resulting extract, the pH was brought to 8. 5 to 9. 0 with 1M NaOH by using pH meter. Then, mixed gently and centrifuged at 12,000 x g for 10 minutes. 3. The supernatant were removed and placed into a 50ml beaker. (1ml of supernatant was kept as before and labelled it S3). 4. Next, 3M HCL was added drop by drop until the pH reaches 3. by using pH meter. The solution was stirred gently and centrifuged at 12,000 x g for 10minutes.
5. The precipitate was then dissolved with a minimum amount of 50mM ammonium bicarbonate. This final extract was labelled as S4. 6. The amount of protein was estimated using the Bradford assay at 595nm for each step from S1 to S4. The samples were diluted in order to obtain a proper reading within the standard curve. 7. The standard curve was prepared as follows: Tube |1 |2 |3 |4 |5 |6 | |Protein standard (BSA) 100µg/ml (µl) |- |25 |50 |75 |100 |200 | |Distilled water (µl) |200 |175 |150 |125 |100 |0 | |Bradford reagent (ml) |0. |0. 8 |0. 8 |0. 8 |0. 8 |0. 8 | | 8. Samples were measured at 595nm and standard curve was plotted. The concentration of protein in sample S1 to S4 was estimated in µg/ml. Result: Tube |1 |2 |3 |4 |5 |6 |S1 |S2 |S3 |S4 | |Absorbance |0. 000 |0. 102 |0. 137 |0. 222 |0. 335 |0. 430 |0. 216 |0. 493 |0. 294 |0. 125 | | * The absorbance of S1 to S4 gained after series of dilution. Calculation: From the standard curve concentration (µg/µl) of protein converted to µg/ml: (i) S1 = 75 x 10 = 750µg/ml (ii) S2 = 167. 5 x 10 = 1675µg/ml (iii) S3 = 98 x 10 = 980µg/ml iv) S4 = 42. 5 x 10 = 425µg/ml Discussion: The Bradford assay is the most commonly used protein assay because of its accuracy, sensitivity, and convenience. The Bradford assay is much more accurate than A280 measurements, few other substances interfere with its accuracy, it is sensitive to protein concentrations as low as 1 ?g/mL, and it is very rapid, producing a colour change within five minutes. The Bradford assay is based on the binding of Coomassie Blue dye to proteins. The dye alone has a brownish colour and absorbs light most efficiently at 465 nm.
When the dye combines with protein, however, the resulting protein-dye complex has an intense blue colour whose maximum absorption is at 595 nm. The Bradford assay is faster, involves fewer mixing steps, does not require heating, and gives a more stable colorimetric response than other assays. The darker the blue colour of the solution, the higher the protein concentration. The S1 sample was diluted 1:100, S2 diluted 1:200, S3 diluted 1:100 and S4 diluted 1:100. Questions: 1. The Bradford assay is linear over a short range, typically from 0 µg/ml to 2000 µg/ml, often making dilutions of a sample necessary before analysis.
It is also inhibited by the presence of detergents. Much of the non-linearity stems from the equilibrium between two different forms of the dye which is perturbed by adding the protein. The Bradford assay linearizes by measuring the ratio of the absorbances, 595 over 450 nm. 2. An alternative method to measure protein concentration is immunoelectrophoresis. Conclusion: Alpha lactalbumin was isolated and purified from fresh milk using Bradford assay technique. The concentration of protein in unknown sample obtained from standard curve.