Is membrane protein always insoluble? - (Mar/22/2006 )
I overexpressed a transmembrane protein with a His tag in HEK293 cell. Then I use native lysis solution which doesn't contain reducing or denaturing agent. Then I found the protein was in supernant as detected by westernblot. However, the protein seems not bind beads because the protein was detected in lysis solution, washing solution but not in final elution solution.
How to interpret this results? I think the protein is soluble, but His tag may be buried in the protein.
I agree, that's possible.
It already happened.
If possible, put the tag on the other side, or add a linker.
sorry, I don't have much more experience
I agree with Missele. It is very possible for a his-tag to be buried within a protein, or to be otherwise occupied by the protein and unable to interact with an affinity column. I have had personal experience with this.
There are three solutions I can think of. One is to try to purify it without the benefit of an affinity column, which would entail a fairly long process of optimization. Two - put it on the other terminus, like Missele said. Three - if your protein is stable enough, you can unfold it with guanidine hydrochloride, purify it via a column, and then dialyze the pure protein gradually to remove the guanidine.
Whether your protein can survive such treatment and refold to native state is anyone's guess, though.