Each kit contains 10 units of biotinylated thrombin for cleavage of up to 10 mg recombinant protein along with 2 ml of 10X thrombin cleavage buffer and 10 ug of cleavage control protein. Cleavage control protein is converted from a single 67 kDa band to two bands of 39 kDa and 28 kDa following thrombin cleavage. Store all components at -20 C.
Biotinylated thrombin is useful for cleavage of proteins and peptides at the enzyme recognition sequence Leu-Val-Pro-Arg-||-Gly-Ser. Biotinylated thrombin is then easily removed from the reaction with immobilized avidin agarose resin. This preparation is >95% pure on SDS-PAGE and is tested for activity using thrombin chromogenic substrate and cleavage control protein.

Biotinylated Thrombin Cleavage Protocol
1. Combine 100ul of 10X cleavage buffer, 1mg of target protein, and 1U of biotinylated thrombin with ultrapure water to a total volume of 1ml.
2. Monitor cleavage with a parallel reaction of cleavage control protein in the same buffer as the target protein.
3. Incubate overnight at room temperature.
4. Remove biotinylated thrombin with immobilized avidin or streptavidin agarose resin according to the manufacturer's recommended directions. This resin cannot be reused.
5. Residual uncleaved fusion tagged protein along with cleaved purification tag in the reaction can usually be removed by reapplying to the original purification resin after reequilibration in binding buffer.

Notes: Optimization of biotinylated thrombin protocol is required for specific applications. Recommended reaction conditions are pH 7.0 to 9.0 at 20C. The reaction rate will be increased by increasing the enzyme to protein substrate ratio and incubation temperature. For example, addition of 2U of biotinylated thrombin and incubation at 37C will result in accelerated thrombin digestion (30-60 minutes).
Cleavage at sites similar to the enzyme recognition sequence can be limited by decreasing enzyme to protein substrate ratio and incubation temperature. A pilot scale digestion is recommended for optimization of the reaction by removing samples at 2, 4, 8, and 16 hours and analyzing by SDS-PAGE.
Biotinylated thrombin can be diluted in TBS or PBS.