HARVEST PROTOCOL TO REDUCE VARIABILITY OF SOLUBLE ENZYME YIELD FROM CULTURED-CELLS

Many aspects of physiology and gene regulation can be studied by exami ning the levels of enzymes harvested from cultured cells. We found tha t the yield from cultured cells of two different cytosolic enzymes, cr eatine kinase and the common reporter gene product chloramphenicol ace tyltransferase (CAT), could be highly variable despite superficially i dentical harvest procedures. Analysis of multiple harvest and assay pa rameters disclosed that fluctuations in enzyme yield were correlated w ith the rime cells that were allowed to remain in an EDTA-containing b uffered saline solution prior to scraping from the dishes with a rubbe r policeman. The highest and most consistent yields were obtained when the cells were allowed to remain in the solution for 6-10 min before scraping; this protocol has cut variability approximately by a factor of three.