Question

What column would you use to separate a protein with a pI of 5.8 from one at 7.3? ________________

In your column, what pH needed to separate the two proteins? __________________

Which protein comes off first from the column? _____________________________________

Why?_________________________________________________________________________________

Answer 1

Most proteins are separated with a Sephadex column. The buffers that you would use would be different to elute out the protein from the column. If an acidic buffer is used

with a pH < than 7 then the protein with pI 7.3 will be protonated and a cation exchange column can be used .  

For a protein with pI - 5.8 it is already in the protonated form so it can be eluted out on a cation exchange column.  

To separate it from the protein with pI= 7.3 by using a salt at pH 7 to separate it from the column as at pI 7.3 it is pretty neutral.

The protein at pI= 7.3 will be eluted out first as it has almost no charge therefore it will not be retained by the column.