Immobilized bovine thrombin is ideal for cleavage of recombinant fusion proteins with an Leu-Val-Pro-Arg/Gly-Ser thrombin recognition sequence. The resin is easily removed by centrifugation or filtration following the reaction. Immobilized bovine thrombin is highly specific, useful in a wide range of reaction buffers and temperatures, and reusable.

Immobilized Thrombin Digestion Protocol
1. Make a digestion buffer consisting of 0.05M Tris-HCl, 0.01M CaCl2, pH 8.0.
2. Dissolve 1 mg of your protein sample in 1 ml digestion buffer.
3. Wash 0.05-0.1ml of immobilized thrombin 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 thrombin gel in ~0.2ml of digestion buffer then add the resin to your protein sample.
5. Incubate mixture in a shaking water bath for 1-2 hours at 37C, or overnight at 4C, with rapid agitation.
6. Separate the immobilized thrombin gel from the digestion mixture as noted in step 3. Retain supernatant as your thrombin-digested protein sample.

Note: Optimization of immobilized thrombin protocol is required for specific applications. Recommended reaction conditions are pH 7.0 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 thrombin digestion (30-60 minutes).