MCE TLCK-treated Chymotrypsin is a high-purity serine protease widely used in protein digestion workflows, enzymatic cleavage studies, proteomics sample preparation, and biochemical research. Chymotrypsin specifically cleaves peptide bonds on the carboxyl side of aromatic amino acids, such as phenylalanine, tyrosine, and tryptophan.

This preparation is treated with TLCK (Nα-p-Tosyl-L-lysine chloromethyl ketone), a selective trypsin inhibitor, to eliminate residual trypsin activity and ensure highly specific chymotryptic digestion, making it ideal for applications requiring strict control of cleavage patterns.

Key Features

• Highly purified serine protease with defined substrate specificity
• TLCK-treated to inactivate trace trypsin contamination
• Reliable cleavage at aromatic residue sites
• Suitable for proteomics, peptide mapping, and structural biology
• Available in multiple convenient pack sizes (5–100 mg)
• Stable enzyme activity with ≥45 U/mg protein
• Supplied as a white to off-white solid
• For research use only

Biochemical Characteristics

Recommended Use and Methodology

Enzyme Reconstitution

• Dissolve TLCK-treated Chymotrypsin in 1 mM HCl
• Recommended working concentration: 0.5–1 µg/µL
• Add 2 mM CaCl₂ as a stabilizer
• Resuspended enzyme can be stored up to one week at 4°C

Protein Digestion Protocol

1. Protein Solubilization / Denaturation

• Dissolve target protein in 100 mM Tris-HCl, 10 mM CaCl₂ (pH 8.0)
• Difficult proteins may require 6–8 M urea or 6 M guanidine HCl
• Optional: Heat sample at 60°C for 1 hour or 95°C for 15–20 minutes for resistant proteins

2. Reduction of Disulfide Bonds

• Add 5 mM DTT or β-mercaptoethanol
• Incubate at 50–60°C for 20 minutes

3. Alkylation

• Cool to room temperature
• Add iodoacetamide (15 mM)
• Incubate 15 minutes in the dark

4. Buffer Adjustment

• Dilute with Tris-HCl/CaCl₂ buffer until urea/guanidine ≤1 M

5. Digestion

• Add TLCK-treated Chymotrypsin at a 1:200 to 1:20 protease:protein ratio (w/w)
• Incubation: 2–18 hours at 25°C
• Stop reaction by adding 0.5% TFA

Applications

• Proteomics sample preparation
• Peptide mapping and sequence analysis
• Protein structural and folding studies
• Enzymatic cleavage assays
• Preparation of peptide fragments for mass spectrometry
• Digest optimization workflows for biochemical research

Storage & Handling

• Store dry enzyme at recommended conditions (refer to supplier’s specifications)
• Avoid repeated freeze–thaw cycles
• Use sterile, protease-free tools and solutions for handling

MCE TLCK-treated Chymotrypsin provides a highly controlled and specific proteolytic tool for researchers requiring accurate cleavage of proteins at aromatic residues. The removal of residual trypsin activity through TLCK treatment ensures superior specificity, making this enzyme exceptionally suited for proteomics, enzymatic digestion protocols, and protein structure investigations. Its strong performance, versatility, and detailed preparation workflow make it an essential reagent for advanced biochemical and analytical laboratories.