Question

11. From the table in question 10, describe a general technique for separating histidine from a mixture of His, Tyr, and Arg using an anion exchange media. At what pH would you equilibrate the column? At what pH would each amino acid elute? charged group 12. Compare the dye-binding protein concentration assays, Bradford and BCA in terms of accuracy and ease-of-use. The dyes bind to different areas of the protein. How does this impact their accuracy? How can you improve their accuracy?

Answer 1

Answer

11. PI of histidine, tyrosine, and arginine are 7.64, 5.63 and 10.76 respectively so if equilibration buffer of ph=8 would give a mild negative charge on histidine, strong negative charge on tyrosine and positive charge arginine.

Therefore, in anion exchanger column arginine will not bind at ph=8 and washed off in the washing step , then lesser salt concentration in elution buffer will elute the histidine first then later strong salt elution buffer elute the tyrosine.