Proteases:

Our proteases are tested for activity using both peptide substrates and, where appropriate, a protein containing the cognate site. Contamination by non-specific protease activity is assayed by incubation with a standard mixture of proteins for 20 hours at 37°C in a total reaction volume of 10 µl. Results are visualized by SDS-PAGE and Coomassie Blue staining or MALDI-TOF MS where appropriate.

Glycosidases:

All of our glycosidases are tested for contaminating proteases and glycosidases. Since p-nitrophenyl-glycosides are not hydrolyzed by some exoglycosidases, we use both p-nitrophenyl-glycosides and fluorescently-labeled oligosaccharides to screen for contaminating glycosidases.

Assay for Protease Contamination
Contaminating protease activity is typically assayed by incubating a 5- to 10-fold excess of each enzyme with a standard mixture of proteins for 20 hours at 37°C in a total reaction volume of 10 µl. Results are visualized by SDS-PAGE and Coomassie Blue staining.

Assay for Contaminating Exoglycosidases and Endoglycosidases
PNP-Glycoside Assay:
A 5- to 10-fold excess of each enzyme is incubated with a wide range of p-nitophenyl-glycoside substrates at 10 mM concentration, in a 50 µl reaction, at 37°C for 20 hours.

AMC-labeled Oligosaccharide Assay:
(AMC=7-amino-4-methyl-coumarin)A 5- to 10-fold excess of each enzyme is incubated with 1 nmol of a wide range of AMC-labeled oligosaccharides, in a 10 µl reaction at 37°C for 20 hours.

Protein Kinases and Phosphatases:

Assay for Protein Phosphatase Contamination
Each purified protein phosphatase is rigorously tested for the presence of contaminating protein phosphatases by measuring the release of radioactive inorganic phosphate from appropriate phosphory-lated peptides or proteins. Thus, tyrosine-specific protein phosphatases are tested for the presence of contaminating serine/threonine-specific phos-phatases and vice versa. Nonspecific protein phosphatase activity is measured using the PNPP assay described below. Typically, the normal reaction conditions are employed with a 10- to 20-fold excess of the protein phosphatase. Each purified protein kinase is rigorously tested for the presence of contaminating protein phosphatase activity by spectrophotometric analysis of the hydrolysis of p-nitrophenylphosphate (PNPP).

Assay for Protease Contamination
Contaminating protease activity is typically assayed by incubating a 10- to 20-fold excess of each enzyme with a standard mixture of proteins for 2 hours at 30°C. Results are visualized by SDS-PAGE and Coomassie Blue staining.