Thrombin Sepharose beads are used for efficient and convenient cleavage of recombinant fusion proteins containing thrombin-specific cleavage site.
- Efficient and convenient cleavage of recombinant fusion proteins
- Formulation is 6% cross linked Sepharose beads provided as 50% slurry in pure glycerol
- Maximize the recovery of the target protein and its cleaved fragment
Thrombin Sepharose beads for efficient and convenient cleavage of recombinant fusion proteins containing thrombin-specific cleavage site
In order to find the optimum cleavage conditions for a target fusion protein, it is recommended to run preliminary cleavage reactions at a small scale. Successful cleavage with thrombin is dependent upon proper folding of the fusion protein that enables access of the thrombin recognition sequence by the enzyme. Once optimum cleavage conditions are obtained, the reaction can be scaled up to cleave the entire amount of the target fusion protein. The target fusion protein should be purified to homogeneity and dialyzed against 50 mM Tris buffer, 0.1 M NaCl, pH 8.0 before setting up the cleavage reaction.
To purify the target protein, re-suspend the beads by gentle swirling. Do not Vortex. Then, aliquot 15 µl of the suspended slurry and add to 1 mg of the fusion protein in an Eppendorf tube. The recommended concentration of target fusion protein is 1 mg/ml. Mix gently by inverting the tube (do not vortex) and gently shake on a rotary shaker at room temperature. At regular time intervals spin down the tube to aliquot a test sample and freeze it immediately. At the end of the reaction, analyze the samples by SDS-PAGE.
To recover the cleaved target protein, after the fusion protein is completely cleaved, spin down the reaction mixture for two to three minutes at 5000 rpm. Then, remove the supernatant and wash the beads with 0.2 ml of 50 mM Tris buffer, pH 8. Repeat these steps to maximize the recovery of the target protein and its cleaved fragment. Further chromatography may be necessary to remove the cleaved fragments from the target protein.
