TLCK-treated Chymotrypsin is a type of serine protease that cleaves protein chains at the carboxyl side of aromatic amino acids. It undergoes TLCK treatment to inactivate residual trypsin activity.

Prepare a chemical compound with a concentration of 10 mg/mL by using 1 mM HCl, and include 2 mM calcium chloride as a stabilizer. Store the prepared solution at 4℃ for up to one week. For protein preparation, dissolve and denature proteins in 100 mM Tris-HCl, 10 mM CaCl2 (pH 8.0). Proteins that are difficult to dissolve or require denaturation for effective digestion can be dissolved at room temperature to 37℃ in minimal volume with denaturants such as 6-8 M urea or 6 M guanidine hydrochloride for 1 hour. Some proteins may benefit from heating the sample to 60℃ for 15-20 minutes to aid solubilization and denaturation. For disulfide bond reduction, add DTT or β-mercaptoethanol to the dissolved protein to achieve a final concentration of 5 mM, and heat the sample at 50-60℃ for 20 minutes. Alkylation involves allowing the reduced protein mixture to cool to room temperature before adding iodoacetamide to a final concentration of 15 mM. Finally, adjust the reaction volume to ensure urea or guanidine hydrochloride is less than or equal to 1 M with 100 mM Tris-HCl, 10 mM CaCl2 (pH 8.0). For enzyme reconstitution, dissolve chymotrypsin in 1 mM HCl with a recommended concentration of 0.5-1 μg/μL. For digestion, add chymotrypsin to achieve a final protease-to-protein ratio of 1:200 to 1:20 (w/w) and incubate the sample at 25℃ for 2-18 hours. Terminate the digestion reaction by adding 0.5% trifluoroacetic acid if needed.