how do GST-tags affect gel filtration results?
Posted 12 September 2012 - 08:23 AM
Posted 12 September 2012 - 12:44 PM
BTW: as far as I know if a protein is soluble only with GST and precipitates when you cut the GST, then that protein was not soluble in the first place i.e. was not folded in the cell. Are you sure your protein is usable at all? Not that you work to purify it for nothing.
Posted 12 September 2012 - 01:24 PM
I have been facing some issues with the protein's activity. My initial suspect was that the GST-tag was interfering with the assay and so I had tried Factor Xa to cleave it off. I still do not see any activity. Literature shows that it tends to show a very weak nucleosidase activity but the assays done in the paper do not include Factor Xa.
So I am quite clueless right now.
also, I am not entirely sure as to what kind of readout should I expect with ion exchange as I am not really trying to purify the protein further but trying to see if it form higher order oligomers instead.
Posted 12 September 2012 - 01:33 PM
I am not really trying to purify the protein further but trying to see if it form higher order oligomers instead.
In my opinion, as long as you have GST there you cannot do it.
also if you don't get activity + your protein precipitates after you cleave the tag, be assured that it was born dead. Try different expression conditions aka make an expression test/screen based on activity. It is so nice to work with enzymes that have an activity to check. I would take different expression conditions: temperatures, times, media, inducer concentration and harvest 10 mL culture for each of them, resuspend in 2 mL buffer and sonicate after which spin down to clear the lysate and check some of the lysate for activity. If you don't have activity in the cell lysate, the protein is not folded so don't bother purify it. Just my two cents.
Posted 12 September 2012 - 03:44 PM
However, with all of these I did not check the activity of the protein.
Posted 12 September 2012 - 09:39 PM