You have been tasked with purifying Protein A Previous exper

You have been tasked with purifying Protein A. Previous experiments have determined the following information about Protein A and the contaminating proteins (B, C, and D). Beginning with a mixture of all four proteins, design a purification scheme to separate Protein A from the contaminating proteins. For chromatography steps, indicate which elution fractions (early vs. late) will contain which protein(s). A colleague has developed an alternative purification method, but it ends with Protein A in a solution of 8 M urea, and the protein is not functional. What has happened to the protein? What specific purification method could you use to restore the function?

Solution

We will use Ion-Exchange Chromatography, in which we use cation exchanger because proteins are majorly positive. During that first less basic protein will elute out and then more basic protein. So we find the proteins in the order B> C> A. D will remain in the column because it has negative charge.

After using more than 1M salt or urea protein, get nonfunctional. Urea denatures protein by binding to hydrophilic regions like amide with hydrogen bonds. We use ice-cold ethanol in 9:1 ratio to remove excess urea.