V8 protease belongs to the serine protease family and specifically hydrolyzes the peptide bonds on the carboxyl side of glutamic acid (Glu, E) or aspartic acid (Asp, D) residues. It recognizes and cleaves the carboxyl-terminal peptide bonds of Glu in NH₄HCO₃ buffer (pH 7.8) and CH₃COONH₄ buffer (pH 4.0). In phosphate buffer (pH 7.8), it recognizes and cleaves the carboxyl-terminal peptide bonds of both Glu and Asp, with a higher hydrolysis rate for Glu than for Asp.
Product Applications
When used in protein digestion for sequencing, peptide mapping analysis, and peptide mass fingerprinting analysis, it can be used alone or mixed with other proteases.
Basic Properties
V8 protease is active in the pH range of 4.0–10.0, with an optimal pH of 8.0–8.5.
Inhibitors of V8 protease include diisopropyl fluorophosphate (DFP), α₂-macroglobulin, and Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK).
Usage Method
Precautions before use: Aliquot according to the required amount to minimize contamination or autolysis.
Dissolution of target protein: Dissolve the target protein in digestion buffer (e.g., 25–50 mM NH₄HCO₃, pH 7.8). If solubility is poor, denature the target protein by adding urea, SDS, DTT, or heating. The effects of urea and SDS on V8 protease are shown in Table 1.
Digestion: The recommended mass ratio of V8 protease to target protein is 1:20–1:100 (W/W). Perform digestion at 25°C or 37°C for 2–18 hours.
Table 1: Stability of V8 protease in the presence of denaturants at different concentrations (urea: 25°C for 2 hours; SDS: 25°C for 12 hours)
(Note: V8 protease concentration: 0.5 mg/ml; buffer: 50 mM Tris-HCl, pH 8.0)
Application Example: Digestion of Insulin with Recombinant V8 Protease
Mix 100 μl insulin solution, 198 μl 0.4 M Tris-HCl (pH 8.0), 60 μl 0.1% V8 protease solution, and make up to 400 μl with ultrapure water. Incubate in a water bath at 37°C for 2 hours, then add 6 μl phosphoric acid solution to terminate the reaction. Analyze using a C18 column.
Storage
Storage stability: Lyophilized recombinant V8 protease is stable for 24 months when stored at 2–8°C.
- After reconstitution in water or 50 mM Tris-HCl (pH 8.0), it is stable for 6 months when stored below -20°C at a concentration of 1 mg/ml.
- After reconstitution in water or 50 mM Tris-HCl (pH 8.0), it is stable for 2 months when stored at 4°C at a concentration of 1 mg/ml (aseptic condition).
- After reconstitution in 50 mM Tris-HCl (pH 8.0), it retains over 85% of its activity for 12 hours when stored at 25°C or 37°C at a concentration of 0.5 mg/ml.
- The reconstituted solution can withstand 10 freeze-thaw cycles without any loss of activity.
Product Advantages
Sequence-grade trypsin (analytical pure): High specificity and good stability.
Animal-origin-free: Produced by recombination, no exogenous virus contamination, and no animal-derived raw materials used in the production process.
Stable quality: Manufactured in batches to ensure stable and continuous production; no variation between batches, with consistent quality.
High purity: High specific activity; residual host protein is below the limit required for biological products.
Lyophilized powder: Easy to store and transport.
Compliance with regulatory requirements: Production equipment and environment meet relevant regulatory requirements; the production process fully complies with the NSF ISO 9001:2015 quality system and GMP guidelines.
Complete quality documents: Relevant regulatory support documents can be provided upon customer request.
