Harvest protocol to reduce variability of soluble enzyme yield from cultured cells.
Many aspects of physiology and gene regulation can be studied by examining the levels of enzymes harvested from cultured cells. We found that the yield from cultured cells of two different cytosolic enzymes, creatine kinase and the common reporter gene product chloramphenicol acetyltransferase (CAT), could be highly variable despite superficially identical harvest procedures. Analysis of multiple harvest and assay parameters disclosed that fluctuations in enzyme yield were correlated with the time cells that were allowed to remain in an EDTA-containing buffered saline solution prior to scraping from the dishes with a rubber 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.
Buskin
J N
JN
University of Washington, Seattle 98195-7350, USA. buskin@u.washington.edu
Gregory
D L
DL
LaFramboise
W A
WA
Hauschka
S D
SD
D000818:Animals; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000302:isolation & purification*; D003402:Creatine Kinase / Q000302:isolation & purification*; D005069:Evaluation Studies as Topic; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D051379:Mice; D009132:Muscles / Q000166:cytology / Q000201:enzymology; D015203:Reproducibility of Results; D012965:Sodium Chloride; D012995:Solubility; D012996:Solutions; D014162:Transfection
D012996:Solutions; D012965:Sodium Chloride; D015500:Chloramphenicol O-Acetyltransferase; D003402:Creatine Kinase