TLCK-treated Chymotrypsin

Recommended to reconstitute in 1mM HCl at 10 mg/mL and add 2 mM calcium chloride as stabilizer. Resuspended Chymotrypsin can be stored for up to one week at 4°C
Preparation of Protein
1. Solubilization/Denaturation: Dissolve protein in 100 mM Tris-HCl, 10 mM CaCl2 (pH 8.0). Proteins that are difficult to dissolve or require denaturation for efficient digestion can be solubilized in a minimum volume in a denaturant such as 6–8 M urea or 6 M guanidine HCl at room temperature to 37°C for up to one hour. For some proteins, it may be beneficial to heat the sample to 60°C over this time period (95°C for 15–20 minutes for extreme cases).
2. Disulphide Reduction: To the dissolved protein add DTT (or β-mercaptoethanol) to a final concentration of 5mM; heat this sample at 50–60°C for 20 minutes.
3. Alkylation: Allow the reduced protein mixture to cool to room temperature, and add iodoacetamide to a final concentration of 15 mM. Incubate in the dark for 15 minutes at room temperature.
4. Finally adjust the reaction volume with 100 mM Tris-HCl, 10 mM CaCl2 (pH 8.0) such that the urea or guanidine concentration is 1 M or less.

Enzyme Reconstitution
Dissolve Chymotrypsin in 1mM HCl. We recommend a final concentration 0.5–1 μg/μl.

Digestion
Add Chymotrypsin to a final protease:protein ratio of 1:200 to 1:20 (w/w), and incubate sample for 2–18 hours at 25°C. The reaction may be stopped, if desired, by adding 0.5% trifluoroacetic acid.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.