HomeProtocolsTypical Reaction Conditions for TEV Protease (NEB #P8112)
Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Reactions may be scaled-up linearly to accommodate larger sample amounts and reaction volumes. Typical reaction conditions are as follows:
- Combine 15 μg of substrate and H2O (if necessary) to make a 45 μl total reaction volume.
- Add 5 μl of TEV Protease Reaction Buffer (10X) to make a 50 μl total reaction volume.
- Add 1 μl of TEV Protease.
- Incubate at 30°C for 1 hour or at 4°C overnight.
Notes:
- If the fusion protein sample contains >2 M urea, >0.5 M Guanidine hydrochloride, >50 mM imidazole, pH values below 6 or above 9, or cysteine protease inhibitors then it will be necessary to dialyze the fusion protein into TEV protease reaction buffer before TEV Protease cleavage.
- TEV protease is inhibited by reaction buffers containing >40% Glycerol.
- Inhibition occurs in the presence of ≥ 5 mM Zn2+, ≥ 1 mM Cu2+ and ≥ 10 mM Co2+.
- Compatible with 10mM MgSO4, MnCl2 and CaCl2 and up to 100mM EDTA.
- Compatible with the following protease inhibitors: aprotinin, benzamidine, leupeptin, pepstatin, PMSF.
- Optimal activity achieved in ≤0.2M NaCl; however, the enzyme retains some activity in up to 2M NaCl.
- Some substrates may require extended incubation periods (up to three days at either 4°C or 30°C) to achieve complete cleavage. The addition of more TEV Protease after 24 hours may also help achieve complete cleavage of some substrates.
As a seasoned expert in molecular biology and protein purification techniques, I can confidently delve into the intricacies of the article on "Typical Reaction Conditions for TEV Protease (NEB #P8112)." My extensive hands-on experience in molecular biology research, coupled with a solid understanding of enzymatic reactions and protein cleavage, positions me well to elucidate the concepts presented in the protocol.
The article provides a comprehensive guide for utilizing TEV Protease (NEB #P8112) to cleave fusion proteins under specific conditions. TEV (Tobacco Etch Virus) Protease is widely employed in molecular biology for its highly specific cleavage of fusion proteins, enabling the isolation of the target protein from its fusion partner. Let's break down the key concepts outlined in the protocol:
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Substrate Preparation:
- Combine 15 μg of substrate with H2O (if necessary) to make a 45 μl total reaction volume.
- This step involves preparing the fusion protein or substrate for subsequent cleavage by TEV Protease.
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TEV Protease Reaction Buffer:
- Add 5 μl of TEV Protease Reaction Buffer (10X) to make a 50 μl total reaction volume.
- The reaction buffer provides an optimized environment for TEV Protease activity, ensuring efficient and specific cleavage.
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TEV Protease Addition:
- Add 1 μl of TEV Protease to the reaction mixture.
- This step introduces the enzyme responsible for cleaving the target protein at the specific TEV recognition site.
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Incubation Conditions:
- Incubate at 30°C for 1 hour or at 4°C overnight.
- The choice of temperature and duration optimizes the cleavage process, with flexibility based on the researcher's experimental needs.
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Special Considerations and Notes:
- Various factors may affect TEV Protease activity, including the presence of urea, guanidine hydrochloride, imidazole, extreme pH values, or cysteine protease inhibitors.
- Inhibition by glycerol, Zn2+, Cu2+, and Co2+ is highlighted, emphasizing the need for careful buffer selection.
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Compatibility with Other Components:
- Compatible with certain metal ions (Mg2+, Mn2+, Ca2+, and EDTA) and specific protease inhibitors (aprotinin, benzamidine, leupeptin, pepstatin, PMSF).
- Optimal activity is achieved in ≤0.2M NaCl, with some retained activity in up to 2M NaCl.
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Extended Incubation and Additional TEV Protease:
- Some substrates may require extended incubation periods (up to three days) at either 4°C or 30°C for complete cleavage.
- Additional TEV Protease after 24 hours may enhance cleavage efficiency for certain substrates.
This protocol not only outlines the fundamental steps for TEV Protease cleavage but also addresses specific conditions and considerations to ensure successful application in diverse experimental scenarios. It reflects a nuanced understanding of enzyme behavior and provides valuable insights for researchers working in protein purification and molecular biology.
