I am treating cells with all kinds of compounds, and hope to detect protein X enzymatic activity with cell lysate. But protein X has many family members (X, X1, X2, X3,.....Xn) which use same/similar substrate and I cannot distinguish X's activity from Xn's activity, so I have to isolate (and maybe purify) protein X to do activity assay.
Any good ideas? Really appreciate it!
I agree that purifying the enzyme would be a very difficult and time consuming task. Rather, you can use a crude fractionation method to enrich the protein of interest. If X differs significantly from X1, X2, X3...Xn in some physicochemical parameter like charge, mass or hydrophobicity, you can use a quick incubation with ion-exchange beads, size fractionation or ammonium sulphate cut. A slightly tedious way would be to pass the lysate through a column charged with the antibody for X ( an affinity column), discard the flow-through, before assaying the activity of the elute.
Thank you so much, Bharath. The second method looks possible. Actually I thought about that strategy, but the question is that how to separate protein X from antibody, and that the antibody MAY block the enzymatic activity by binding to the activity domain.
that is fairly simple, elute the protein with a low pH or high salt buffer since bulk of the specific interactions between the antibody and your protein of interest are enthalpic in nature...you will get a reasonably pure protein X eluting out of your column...
WOW, I think you must be good at this tech. Could you please give me some papers? I need to know detailed protocols. Thank you so much.
I am attaching this techtip from Thermo. Any paper searched with the term "immunoaffinity protein purification" will give you such protocols as the attached document gives. Best Wishes!
Thank you so much. Let me read it. If more questions I will ask you for help.
Alternatively, if you can get hold of an antibody that binds your enzyme but does not affect its activity, you may coat a microplate with said antibody and pull down your enzyme of interest from the lysate. Allows parallel determination with lots of sample.
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