Immobilized bovine chymotrypsin is ideal for digestions of proteins and peptides. Chymotrypsin is a protease that cleaves peptide bonds at Trp, Tyr, Phe, Leu and Met. Chymotrypsin protease is generally specific for hydrophobic residues and is often used to provide complementary sequence coverage when compared to tryptic digested samples.

Immobilized Chymotrypsin Digestion Protocol
1. Make a digestion buffer consisting of 0.08M Tris-HCl, 0.1M Calcium Chloride, pH 7.8 (or other suitable buffer such as 0.1M Ammonium Bicarbonate, 0.01M Calcium Chloride, pH 8.0).
2. Dissolve 1 mg of your protein sample in 0.2ml digestion buffer.
3. Wash 0.2-0.3ml of immobilized chymotrypsin 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 chymotrypsin 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 chymotrypsin gel from the digestion mixture as noted in step 3. Retain supernatant as your chymotrypsin-digested protein sample.

Note: Optimization of immobilized chymotrypsin 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 chymotrypsin digestion (30-60 minutes).