UniHRV 3C Protease
UniHRV 3C Protease is a recombinant form of the 3C protease derived from human rhinovirus 14 expressed in E. coli (specific activity 1800 2000 U/mg).
This product is a highly purified recombinant 6XHis fusi on protein and requires neither metal nor cofactor s for activity. UniHRV 3C Protease recognizes the cleavage site: Leu Glu Val Leu Phe Gln↓Gly Pro (LEVLFQ↓GP). UniHRV 3C Protease demonstrate excellent cleavage efficiency in a variety of fusion proteins.
This product is a highly purified recombinant 6XHis fusi on protein and requires neither metal nor cofactor s for activity. UniHRV 3C Protease recognizes the cleavage site: Leu Glu Val Leu Phe Gln↓Gly Pro (LEVLFQ↓GP). UniHRV 3C Protease demonstrate excellent cleavage efficiency in a variety of fusion proteins.
Catalog number:
C09003-bulk / C09003-500U UniHRV 3C Protease
C09002-B-bulk / C09002-B-1 10X HRV 3C Cleavage Buffer
Package:
UniHRV 3C Protease / 500U
Cleavage Validated Protein / 20 μg
10X HRV 3C Cleavage Buffer / 10 mL
Source:
Escherichia coli
Endotoxin level:
<1 EU per 1 μg of the protein by the LAL method.
Activity:
One unit of HRV 3C Protease is defined as the amount of enzyme needed to digest 1 nM of the pNA-peptide substrate per 10 min at RT or per hour at 0 °C in the reaction buffer.
Purity:
>98% as determined by SDS-PAGE.
Formulation:
UniHRV 3C Protease was lyophilized from a solution containing 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.04% Tween20, 8% trehalose, 8% mannitol.
10X HRV 3C Cleavage Buffer: 1.5 M NaCl, 0.5 M Tris-HCl, pH 7.5
Reconstitution:
It is recommended to reconstitute the lyophilized protein in sterile H2O and incubate the stock solution for at least 20 min to ensure sufficient re-dissolved.
Storage:
Lyophilized protein should be stored at -20°C. Upon reconstitution, protein aliquots should be stored at -20°C.
HRV 3C Protease Cleavage Buffer should be stored at -20°C or 4°C.
Shipping Conditions:
Blue ice
C09003-bulk / C09003-500U UniHRV 3C Protease
C09002-B-bulk / C09002-B-1 10X HRV 3C Cleavage Buffer
Package:
UniHRV 3C Protease / 500U
Cleavage Validated Protein / 20 μg
10X HRV 3C Cleavage Buffer / 10 mL
Source:
Escherichia coli
Endotoxin level:
<1 EU per 1 μg of the protein by the LAL method.
Activity:
One unit of HRV 3C Protease is defined as the amount of enzyme needed to digest 1 nM of the pNA-peptide substrate per 10 min at RT or per hour at 0 °C in the reaction buffer.
Purity:
>98% as determined by SDS-PAGE.
Formulation:
UniHRV 3C Protease was lyophilized from a solution containing 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.04% Tween20, 8% trehalose, 8% mannitol.
10X HRV 3C Cleavage Buffer: 1.5 M NaCl, 0.5 M Tris-HCl, pH 7.5
Reconstitution:
It is recommended to reconstitute the lyophilized protein in sterile H2O and incubate the stock solution for at least 20 min to ensure sufficient re-dissolved.
Storage:
Lyophilized protein should be stored at -20°C. Upon reconstitution, protein aliquots should be stored at -20°C.
HRV 3C Protease Cleavage Buffer should be stored at -20°C or 4°C.
Shipping Conditions:
Blue ice
The following protocol is an example of a simple optimization experiment designed to estimate the appropriate ratio of enzyme:target protein. This example represents UniHRV 3C Protease:target protein ratios (U/μg) of 1:10, 1:25 and 1:50.
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Notes (Important):
| Component | Volume (μL) |
| UniHRV 3C Protease (1 U/μL) | 1 |
| Validated Protein (10, 25, and 50 μg each) | X |
| 10X HRV 3C Cleavage Buffer | 5 |
| H2O | 44-X |
| Total volume | 50 |
- Incubate the reaction mixture at 4°C for 16 hours or overnight.
- Determine cleavage level of the samples by SDS-PAGE analysis.
- UniHRV 3C protease: target protein ratio of 1:5~1: 200 (U/μg) is used for most fusion protein cleavage.
- If shorter incubation time is required, more amount of UniHRV 3C protease or higher temperature (RT) can be implemented. Reaction can be performed at 4°C-37°C. 4°C is recommended as the starting standard.
Notes (Important):
- Cleavage efficiency may differ based on structure and properties of each target protein, we recommend testing several enzyme-to-substrate ratios, temperatures, and incubation times.
- HRV 3C Protease reactions can be performed in a buffer which is optimal for the target protein. Reducing reagents (e.g., DTT) or salts (e.g., NaCl) can be added for cleavage efficiency evaluation.
- Usually, 1 U UniHRV 3C Protease can cleavage >95% of target protein (from 50 to 400 μg) at 4˚C for 16h. Even different lot of target proteins might result in different cleavage efficiency. The validated protein (50 μg) provide in this product can be stably cleavage >50% when using 1 U of UniHRV 3C Protease.