A lot of protocols for in-gel trypsin digestion of proteins recommend leaving the dried gel piece to swell with trypsin solution on ice for 15-60 minutes prior to incubation at 37 C. Do anyone know the reasoning behind this pre-incubation on ice?
Christopher S Morello
This step gives time for the enzyme to diffuse into the gel and bind to the protein before the 37 C step can increase the enzyme activity. Without the in-gel protein as the substrate, the trypsin can undergo autolysis and generate extra peptide fragments which can interfere with analysis.
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