Best lysis buffer for mammalian cells to do affinity chromatography? - (Apr/28/2007 )

Hi,

I am trying to find a target protein by using biotin-SA affinity chromatography.
What would be the best lysis buffer condition for mammalian cell (probably use HeLa either suspension or adherent) that does not interfere with affinity chromatography and give most native proteins?
What kind of protease inhibitor do people usually use?

Thanks a lot!

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we use complete from Roche, a pre-mixture of protease inhibitors in tablet form; Roche took over several years ago Boehringer Mannheim which were known for excellent protease inhibitors; they now offer Phosstop, a tablet to stop phosphatase activities;

lysis may succeed but what about homogeneization? is your target part of the plasma membrane, of organelles, of nucleus or of cytoplasm?

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Thanks 'The Bearer'.
Yes, homogeneization is also an issue. It's pretty much a blind search. So we don't really know where this target protein is located. Is there any good protocol for whole cell proteins? If not, I may try to run one for membrane and one for cytosolic proteins and etc. What are the good protocols for each?

Thanks so mcuh!

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Does anyone have any experience with any of those lysis kits available in the market such as Roche complete lysis M kit, Sigma's CelLytic M kit, or Qiagen Qproteome Lysis kit? Do they make any difference?

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