Thank you for your suggestions. I will try the expression checks. I had done quite a few of them to get overexpression of soluble protein as I took me a while to figure out the temperature to grow it at (overnight at <18C) and the inducer concentration. Plus, addition of protease inhibitor cocktail (for bacterial cell lysate) and the addition of DTT to the cell lysis buffer helped a lot.
However, with all of these I did not check the activity of the protein.
Thanks a lot !!
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.
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