A radiolabel-release microwell assay for proteolytic enzymes present in cell culture media.
A modified method for the measurement of proteolytic enzyme activity in cell culture-conditioned media has been developed. Using the release of 3H-labeled peptides from 3H-labeled gelatin the method is performed in microwell plates. The substrate is insolubilized and attached to the wells by glutaraldehyde treatment, thus eliminating the need for a precipitation step at the end of the assay. The assay is sensitive, reproducible, and convenient for small sample volumes. The effect of different protease inhibitors on activity can be assessed rapidly allowing an early characterization of the enzyme. It can also be adapted to microplate spectrophotometric analysis by staining residual substrate with Coomassie blue.
Rucklidge
G J
GJ
Biochemistry Division, Rowett Research Institute, Bucksburn, Aberdeen, United Kingdom.
Milne
G
G
D000818:Animals; D003470:Culture Media / Q000378:metabolism; D005780:Gelatin / Q000378:metabolism; D005910:Glioma / Q000473:pathology; D005976:Glutaral / Q000494:pharmacology; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism; D008832:Microchemistry / Q000379:methods; D010447:Peptide Hydrolases / Q000378:metabolism*; D051381:Rats; D012394:Rosaniline Dyes; D013552:Swine; D014316:Tritium; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000201:enzymology
D003470:Culture Media; D012394:Rosaniline Dyes; D014316:Tritium; C048139:Coomassie blue; D005780:Gelatin; D010447:Peptide Hydrolases; D014357:Trypsin; D008666:Metalloendopeptidases; D005976:Glutaral