MadSci Network: Biochemistry

Re: Why there is no lipase activity in my dialysate

Date: Sat May 17 08:53:53 2008
Posted By: Karin Crowhurst, Assistant Professor, Biochemistry
Area of science: Biochemistry
ID: 1210687260.Bc

Hi Sam,

In my understanding, "dialysate" is the stuff that flows through the membrane of the dialysis tubing (and out into bulk solvent). I have to assume you actually mean that you ran the _contents_ of your dialysis tubing on the column, right? Also, if you can't find much activity in your sample before the affinity step, I would assume there would be little activity after that, too. Perhaps I am misunderstanding what you are saying.... but let me provide some information that might be useful:

If your protein is unstable, the problem might be that it is unfolded or misfolded. It might be that the dialysis buffer you are using makes the enzyme more unstable and therefore more unfolded. Unfolded enzymes don't tend to have any enzymatic activity :) Alternatively, your precipitation step might be the culprit. Some proteins just do not deal well with being precipitated out, especially if they are prone to aggregation or unfolding. You might want to think about doing a different first step of your purification (i.e. skipping the salting out step and instead running an extra column in addition to the affinity column). If you want to continue using the salting out procedure, you might want to try re- dissolving the pellet in denaturant (guanidine or urea) and then dialysing the protein in refolding buffer before loading it on your affinity column.

It is possible that none of these things I suggested are the problem, but when dealing with proteins that are a little less stable, sometimes it takes trial and error to get them to cooperate. Some of the "standard" purification methods, such as salting out, that work on robust proteins just don't work on others.

Good luck!

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