Does anyone still do that? I do, and here is what happened. I am trying to enrich an 'activity of interest' from a human cell lysate 1960s style in hopes of identifying the responsible protein. So, first a hard spin in an ultra centrifuge (activity stays in supernatant) then trial ammonium sulfate fractionation. Result:
0% saturation (mock) - no activity in supernatant.
15% saturation - no activity in supernatant.
30% saturation - activity in supernatant!!!
60% saturation - no activity in supernatant.
Any thoughts?? Protein of interest is an RNA binder, ammonium sulfate fractions were buffer exchanged into Tris + 10 mM MgOAc2 and 100 mM KCl before assay.
Thanks. Still seems strange to me that activity would be present in the soluble fraction before salt precipitation and then disappear after mock ammonium sulfate treatment.... only to return at a higher salt concentration. Luckily for me it only has to work once, and a little voodo is ok. Just trying to clean up the lysate as much as I can before I biotinylate the binding partner and use it to hook the mystery protein out for mass spec.
Your task seems to be a little bit tricky - have you checked the acitivity also in precipitate? It is likely that at 60% is your protein completely precipitated and therefore you have no activity in supernatant (?). And maybe you could try another old-style method: cold aceton precipitation. Good luck.
If it works, there is nothing wrong with old techniques. Especially ones as cheap as AS precipitation. Assuming yours is a reproducible result, it suggests that something is happening to your protein during dialysis. The outlier here in the results is the lack of activity at 0% and 15%. The first two things that leap to mind are your protein sticking to the dialysis membrane or alternatively it is being degraded by proteases. And then presumably for your 30% AS cut, you would be inactivating the proteases (precipitation) or preventing your protein from sticking to the membrane. I would need to know more details about sample prep before and during dialysis to comment further.
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