1. In order to prepare the bovine serum albumin (BSA) standard for this assay, it would be necessary to dissolve a sample of BSA in water or in a buffer, and allow the protein time to dissolve, filter or centrifuge the solution (to remove any un-dissolved materials), and finally determine the exact concentration of the protein. UV absorption is a good way to determine the exact concentration. The published extinction coefficient for bovine serum albumin is E2801% = 6.6. The concentration unit % solution) here are a little odd, and dates to the day before the structure and molecular weight of the protein were known. A 1% solution is:
Or put another way a 10 mg/mL solution will have an A280 = 6.6.
Let's say that 5 mL of a BSA solution is prepared, its A280 is read and A280 = 0.82
What is the concentration of the solution in mg/mL?
2. Show how you would dilute the BSA solution made in Problem 2 to make 5 mL of exactly 1.00 mg/mL BSA.
3. Gelatin is an irreversibly hydrolyzed form of collagen that is used in pharmaceutical, cosmetic, and culinary products. The principle amino acids in collagen are glycine, alanine and proline, with small amounts of phenylalanine, traces of tyrosine and no tryptophan. Will UV spectroscopy be a sensitive way to measure concentration of gelatin? Why or why not?
4. If an experimenter found that the Bradford method using dye binding was not suitable for measuring the target protein, and that UV absorption was not sensitive enough, name another method that might be used to measure the protein.