Protocol using Trypsin-ultra™, Mass Spectrometry Grade (P8101)

Trypsin reactions are typically set up with a ratio substrate protein: trypsin in the range of 30-100:1 and incubated, shaking overnight (16 hrs) at 37°C. With that said different proteins are digested at different rates and efficiencies. In many cases, 2-4 hrs at 37°C is sufficient incubation time. Our default approach for in-house proteomics studies, is a shorter reaction time with 100:1 substrate:trypsin ratio. Reactions are immediately analyzed by MS or MS/MS or stored at -20°C for later use.

Example Trypsin-ultraTM reaction (40:1 substrate:trypsin)
5 μl protein sample (about 2 ug)
4.5 μl Milli-QTM water
10 μl 2x Trypsin-ultra reaction buffer
0.5 μl 100 ng/μl Tyrpsin-ultra

Two useful references for trypsin behavior and usage:

Proc., J.L. et al (2010) “A quantitative study of the effects of chaotropic agents, surfactants, and solvents on the digestion efficiency of human plasma proteins by trypsin.” J. Proteome Res. 9, 5422-5437.
Vandermarliere, E. et al (2013) “Getting intimate with trypsin, the leading protease in proteomics.” Mass Spectrometry Reviews 32, 453-465.