For research use only. Not for therapeutic Use.
Chymotrypsin (Chymotrypsin A) is a serine protease produced by the pancreas. Chymotrypsin cleaves protein chains at the carboxyl side of aromatic amino acids[1][2].
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℃.
Preparation of Protein
1. Solubilization/Denaturation: Dissolve protein in 100mM Tris-HCl, 10mM 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 6-8M urea or 6M guanidine HCl at room temperature to 37℃ for up to one hour. For some proteins, it may be beneficial to heat the sample to 60℃ over this time period (95℃ 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℃ for 20 minutes.
3. Alkylation: Allow the reduced protein mixture to cool to room temperature, and add iodoacetamide to a final concentration of 15mM. Incubate in the dark for 15 minutes at room temperature.
4. Finally adjust the reaction volume with 100mM Tris-HCl, 10mM CaCl2 (pH 8.0) such that the urea or guanidine concentration is 1M 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℃. The reaction may be stopped, if desired, by adding 0.5% trifluoroacetic acid.
| Catalog Number | I043599 |
| CAS Number | 9004-07-3 |
| Purity | ≥95% |
| Reference | [1]. W.R. Terra, et al. 4.5 – Biochemistry of Digestion, Editor(s): Lawrence I. Gilbert, Comprehensive Molecular Insect Science, Elsevier, 2005, Pages 171-224. [2]. Steven W. Cotton. Chapter 33 – Evaluation of exocrine pancreatic function, Editor(s): William Clarke, Mark A. Marzinke, Contemporary Practice in Clinical Chemistry (Fourth Edition), Academic Press, 2020, Pages 573-585. |
