How to purify a protein (PI 8.0) using His tag? - (Mar/02/2009 )

Hi, all.
I have expressed a protein in e.coli system and now is purifying it using His tag. However, since the Ni-NTA resin binds His tag usually at pH 8.0, the same as the predicted pI of my protein, I could hardly bind my protein well to the resin. I tried binding buffer with pH 7.3 and pH 9.0 separately, but the results show the major part remained in the after-binding buffer.

p.s. I followed the protocol of Qiagen and the expression vector is pQE 1.

Is there any suggestion? Thanks in ahead.

You tried at pH 8.0 and did not succeed?
Does your protein bind mildly to the column or not?
Have you tried milder washes?

And if nothing works, you may as well try to change the tag for purification.

madrius1 on Mar 2 2009, 10:59 PM said:

Thanks~~

y,I tried pH8.0, very weak binding, and most target proteins precipitated
and, I tried denature buffer and native buffer, both the similar results

There is indeed some binding, but it's too little to the continued work...

yep, maybe I should pay attention to the pI earlier than I chose the His tag....

Hey

You can try talon beads, they work at a pH of 7.2 for sure.

Best
TC

T C on Mar 3 2009, 12:36 PM said:

Thank you:)