Lysyl endopeptidase (Lys-C) is a key proteolytic enzyme known for its exceptional specificity, selectively cleaving peptide bonds at the carboxyl side of lysine (Lys) residues. It is widely used in mass spectrometry (MS)-based protein sequencing and proteomics research. However, traditional Lys-C derived from natural sources often exhibits variability in enzymatic activity and purity, which can limit its application in high-precision studies. To address these challenges, recombinant Lysyl endopeptidase (rLys-C), produced via recombinant expression technology, has emerged as the preferred choice for both scientific research and industrial applications.
Product Overview
The sequencing grade rLys-C provided by MtoZ Biolabs exhibits exceptionally high specificity for cleavage at lysine residues. It demonstrates optimal enzymatic activity within a pH range of 8.0-9.0 and retains its proteolytic capacity even under highly denaturing conditions, such as in the presence of 8 M urea. This makes it particularly suitable for digesting target proteins that are resistant to conventional proteases.
The sequencing grade rLys-C is available in lyophilized powder or frozen liquid form and is supplied with an optimized reconstitution buffer to enhance the stability of the enzyme after reconstitution. rLys-C is ideal for efficient digestion of both single proteins and complex protein mixtures, whether in solution or within gels.
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Product Size |
rLys-C Lyophilized Powder (20 μg) |
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Reconstitution Buffer (0.5 mL) |
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Biological Source |
Recombinant expression in E. coli |
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Storage Condition |
Lyophilized powder: -20 °C; Reconstituted solution: -80 °C |
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Optimal pH |
8.0 |
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Shelf Life |
24 months at -20 °C |
Protocol
1. Enzyme Reconstitution
(1) It is recommended to reconstitute the lyophilized powder using the provided reconstitution buffer or 50 mM acetic acid / 1 mM hydrochloric acid (HPLC grade).
(2) After reconstitution, aliquot and store at -80 °C to avoid repeated freeze-thaw cycles. The liquid formulation is ready-to-use and convenient for immediate application.
2. Recommended Digestion Conditions
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Parameter |
Condition |
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Enzyme-to-Protein Ratio (w/w) |
1:25 ~ 1:100 |
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Optimal pH Range |
8.0 |
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Reaction Temperature |
37℃ |
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Digestion Time |
3 ~ 4 hours (adjust as needed) |
3. Digestion System Compatibility
(1) Maintains stable activity in denaturing systems such as 8 M urea and 4 M guanidine hydrochloride.
(2) Can be combined with trypsin to improve digestion completeness and peptide coverage.
4. Reaction Termination Methods
(1) Store samples at -20 °C or below.
(2) Alternatively, add termination reagents such as 0.1% trifluoroacetic acid (TFA) or 1 mM PMSF to prevent overdigestion.
Features and Benefits
1. Purity
Purity >99.9%, as determined by rLys-C peak area using HPLC at 280 nm.
2. High Specificity
Non-specific cleavage <5%; specifically cleaves at the C-terminal side of lysine residues, delivering a well-defined digestion pattern.
3. Excellent Denaturant Tolerance
Maintains enzymatic activity under denaturing conditions such as 8 M urea.
4. High Purity, Low Autolysis
Sequencing Grade rLys-C minimizes background interference, ensuring clean mass spectrometry profiles.
5. Suitable for Complex Samples
Effective for challenging proteins, including poorly soluble and protease-resistant structural proteins.
6. Flexible Application
Compatible with other proteases (e.g., Trypsin) to enhance proteome coverage in MS analysis.
Applications
1. Proteomics Analysis
Pre-digestion for sample preparation, in-depth protein identification, and enrichment of low-abundance proteins.
2. Structural Biology
Initial cleavage of denatured or structurally stable proteins for conformational studies.
3. Dual-Enzyme Strategy Optimization
Used in combination with Trypsin to enhance the accuracy and coverage of protein identification.
4. Complex Sample Processing
MtoZ Biolabs' sequencing grade rLys-C combines high purity, exceptional specificity, and strong denaturant tolerance, making it an ideal choice for precise and efficient protein pretreatment. Its outstanding performance in complex and denatured samples provides a reliable digestion solution for proteomics research, structural biology investigations, and biopharmaceutical development. For more product details and technical support, please feel free to contact us.
FAQs
Q1: What Is the Source and Key Feature of rLys-C?
A1: rLys-C is derived from Pseudomonas aeruginosa protease IV and recombinantly expressed in E. coli. It specifically cleaves at the carboxyl side of lysine residues.
Q2: Can rLys-C Be Used in Denaturing Conditions?
A2: Yes, rLys-C retains proteolytic activity in strong denaturing conditions such as up to 8M urea and 4M guanidine, making it suitable for processing denatured samples.
Q3: What Is the Difference Between rLys-C and Trypsin?
A3: rLys-C cleaves specifically at lysine residues, whereas Trypsin cleaves at both lysine and arginine. Using both enzymes together can improve digestion efficiency and protein sequence coverage.
Q4: Is rLys-C Suitable for in-Gel Protein Digestion?
A4: Yes, rLys-C is effective for digesting proteins in both gel and solution formats.
Q5: How Can I Enhance Protein Digestion Efficiency with rLys-C?
A5: Ensure complete solubilization of proteins, maintain optimal pH and temperature, and for resistant proteins, use 8M urea to enhance digestion efficiency.
