I have been purifying my protein which is fused to GST. But I find that the majority of the fusion proteins sit happily on the column, even after eluting with different conditions. I have eluted starting with 10mM GSSH and went up to 50 mM GSSH and still found my proteins getting eluted slowly (say 50-100 ug/ ml). I tried incubating the column with high 50 mM GSSH Elution Buffer for 30 min and overnight; the yield certainly increases but not getting out the whole protein.. Again I tried using EB with 140 mM NaCl, 1 mM DTT and close to 1% Triton-x-100 but not end point for my elution. I have collected close to 40 mL eluted fractions and now have 2-3 mg yield in 40 mL.
The previous day I purified GST-tag alone with the same column and I had a complete elution in 5-6 fractions (Final one in 30 mM GSSh EB, No protein).
Could any one suggest any reason for this? Any one has any suggestion to get the fusion protein eluted in a smaller volume?
I can post the next part of the problem, once I have some comments on this one.
I guess possible suggestion includes
(1)As mentioned by you interacting the elution buffer for longer time can increase your yield.You can also pass the column with EB at very slow flow rate slower than what you had used for washes.
(2) Its possible that GST fusion proteins but not GST protein tend to aggregate this can also hinder elution process.Possibly you can play with DTT concentration to reduce these intermolecular interactions. Also try to use 2% N-octyl glucoside to disrupt non specific interaction with the resin.
(3)Try to modify the pH of elution buffer some GST tag proteins tend to elute only at higher pH( 8 to 9).If you want to use high GSH concentration then you have to modify your buffer system otherwise the protein would be eluted in acidic condition.
I guess lyophilization could be an answer for you to concentrate your protein that you have currently obtained.Lyophilization could be followed with overnight dialysis with buffer that you want to dissolve your protein.This was routinely done in my previous work place and its a safe way to store your protein for longtime without losing its activity.
Hope these tips work for you
Good Luck
Hi, I think your protein aggregated and precipitated onto the column. This happens when the fused protein is poorly soluble. So I think your problem is not trying to elute it but trying to improve the buffer condition to improve solubility of the GST-fused protein. good luck
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