Native cow Trypsin protein (ab229834)

SDS-PAGE

Functional Studies

Immobilized Trypsin Digestion Protocol
1. Make a digestion buffer consisting of 0.1M Ammonium Bicarbonate, pH 8.0 (or other suitable buffer such as 0.1M Triethylamine Acetate, pH 8.0).
2. Dissolve 1 mg of your protein sample in 0.5ml digestion buffer.
3. Wash 0.15-0.3ml of immobilized trypsin with 3x0.5ml of digestion buffer. Separate the gel from the buffer after each wash by centrifugation or by column. Discard buffer after each wash.
4. Resuspend the washed immobilized trypsin gel in ~0.2ml of digestion buffer then add the resin to your protein sample.
5. Incubate mixture in a shaking water bath for 2-18 hours at 37C with rapid agitation.
6. Separate the immobilized trypsin gel from the digestion mixture as noted in step 3. Retain supernatant as your trypsin-digested protein sample.

Note: Optimization of immobilized trypsin protocol is required for specific applications. Recommended reaction conditions are pH 7.5 to 9.0 at 37C. The reaction rate will be increased by increasing the enzyme to protein substrate ratio and incubation temperature. For example, a typical digestion of 4 hours to overnight can be achieved using a ratio of 1:25 enzyme to protein substrate, while it is recommended to use 1:10 enzyme to protein substrate for accelerated trypsin digestion (30-60 minutes).