Western Blot Visual Protocol: Phase 1: Sample Preparation 

Please accept our apologies for the delayed response! We don’t work with 24 well plates in the Novus lab, but would recommend starting at 10-20 ul lysis buffer. Scraping with a pipette tip should be suitable. Hope that helps!

+NovusBiologicals hrr

From our experience with adherent cells, you can trypsinize and wash with PBS prior to resuspension in lysis buffer. Since the cells would remain intact, trypsinizing can cause less death compared to scraping and fewer changes in metabolic parameters. However, since trypsin is a protease your assay may be affected especially if you are examining transmembrane or membrane associated proteins. (some information on effects of different detachment procedures can be found here: www.ncbi.nlm.nih.gov/pubmed/20337597) It really boils down to what cells you are using, what proteins you are trying to extract, and the eventually assay you are going to be performing. Hope this helps! Please let us know if we can help with anything else :)

Additionally, I think you were referring to the lysate preps from the attached cells. The short answer is YES, you could trypsinize your cells first and lyse them later (by adding RIPA or another buffer). The reasons that using a lysis buffer directly to the attached cells by investigators are mainly because: 1), to maintain the desired volumes of the lysates; 2), to prevent unneeded/uncontrollable proteolytic digestion; and 3), not to add any non- or low-expressed protein(s) to the lysates of interest. However, if harvesting the cells by trypsinization first, investigators could split the harvested cell for their multiple experiments. Hope this information is helpful!

Two additional possible reasons for investigators to add the lysis buffer directly are to potentially 1), stop all biochemical reactions instantly in cells and 2), save time/steps.