Chymotrypsin is a serine endopeptidase that specifically cleaves the C-terminal peptide bonds of tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp), and leucine (Leu). In addition, it can cleave the C-terminal peptide bonds of methionine (Met), alanine (Ala), aspartic acid (Asp), and glutamic acid (Glu) to a certain extent. Residual trypsin can be inactivated and purified using TLCK (tosyl-lysine chloromethyl ketone). This protease can be activated and stabilized by calcium ions (Ca²⁺).
Based on extensive experience in enzyme preparation development, MtoZ Biolabs has launched a high-purity, sequencing grade chymotrypsin solution. Through comprehensive quality control processes, it provides researchers with stable and reliable enzymatic digestion support, covering all application scenarios from basic research to industrial-scale use.
Product Overview
MtoZ Biolabs' sequencing grade chymotrypsin is produced using advanced preparation techniques, offering a high-purity, high-specificity proteolytic solution. It can be used in combination with Trypsin to further enhance protein identification coverage and achieve more uniform protein digestion. It is suitable for applications such as LC-MS/MS mass spectrometry analysis, antibody drug analysis, and post-translational modification studies.
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Product Number |
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Product Specification |
Chymotrypsin Lyophilized Powder (50 μg) |
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Reconstitution Buffer（0.1 mL） |
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Molecular Weight |
25 kDa |
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Storage Conditions |
Lyophilized powder at -20°C Reconstituted solution at -80°C (stable for 1 week) |
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Optimal pH |
7.8 |
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Shelf Life |
24 months at -20°C |
Protocol
1. Chymotrypsin Reconstitution
(1) It is recommended to reconstitute lyophilized chymotrypsin in 1 mM hydrochloric acid (HCl) containing 2 μM CaCl₂ to ensure enzymatic activity stability.
(2) After reconstitution, aliquot and store at -80°C for long-term storage. Avoid repeated freeze-thaw cycles to prevent loss of enzymatic activity.
2. Digestion Conditions
(1) Applicable pH range: 7.0-8.0
(2) Dosage: Recommended at 1:100-1:50 (w/w)
(3) Temperature: 30°C, typically 4-16 hours (adjust based on experimental needs)
3. Digestion Termination
To terminate digestion and prevent overdigestion, the following methods can be used:
(1) Lower the temperature: Store samples at -20°C or below.
(2) Add termination reagents, such as 0.1% trifluoroacetic acid (TFA) or 1 mM PMSF, to prevent excessive enzymatic digestion.
Features and Benefits
1. High Purity
Optimized purification processes remove non-target protease activities and impurities, ensuring digestion specificity.
2. High Specificity
Specifically cleaves at the C-terminus of aromatic amino acid residues (Y/F/W/L), enhancing peptide resolvability.
3. Low Autolysis
Process optimization reduces interference from self-digested fragments, improving mass spectrometry data accuracy.
4. Excellent Batch Consistency
Strict quality control maintains enzymatic activity variation within <5%, ensuring data comparability across experiments.
5. Broad Compatibility
Suitable for complex samples such as cell lysates, tissue extracts, and recombinant proteins, and compatible with various mass spectrometry platforms including MALDI-TOF, Q-TOF, and Orbitrap.
Applications
1. Proteomics
Enhances protein identification coverage and improves resolution of low-abundance regions.
2. Antibody Structure Analysis
Supports molecular characterization of monoclonal antibodies (mAbs) and verification of antigen-binding sites.
3. Post-Translational Modification (PTM) Studies
Applicable for studies of phosphorylation, glycosylation, and other PTMs.
4. Structural Biology
FAQs
Q1: Can Chymotrypsin Be Used in Combination with Trypsin?
A1: Yes. It is recommended to perform partial digestion with chymotrypsin first (2-4 hours), followed by overnight digestion with Trypsin. This dual-enzyme strategy effectively increases peptide coverage in hydrophobic regions and optimizes LC-MS/MS data quality.
Q2: What Are the Effects of PH Values Outside the Recommended Range?
A2: A pH below 7.0 may reduce chymotrypsin activity, leading to decreased digestion efficiency.A pH above 9.0 may increase the risk of self-digestion, resulting in additional background noise.It is recommended to maintain a stable pH using appropriate buffers (such as Tris-HCl or NH₄HCO₃) to ensure optimal digestion performance.
Q3: How to Handle Samples Containing High Concentrations of Detergents?
A3: It is recommended to appropriately dilute the detergent concentration to minimize its impact on Chymotrypsin activity.Compatible detergents (such as 0.1% NP-40) can be selected to reduce disruption to protein structure and digestion efficiency.If necessary, desalting columns or protein purification methods can be used to remove detergents and improve experimental stability.
Q4: Is Chymotrypsin Suitable for Membrane Protein Digestion?
A4: Yes, it is suitable for membrane protein digestion. It is recommended to pre-treat membrane proteins with SDS, urea, or organic solvents before digestion to enhance chymotrypsin efficiency in hydrophobic environments, thereby achieving higher protein identification coverage.
