New England Biolabs, P8118S, Cold-Active TEV Protease

Related Categories Protein Tools,, Proteases,, Protein Purification Applications Fusion Protein Cleavage,, Protein Analysis Tools Specification Unit Definition One unit of Cold-Active TEV Protease will cleave 2 µg of MBP-fusion protein, MBP5-TEV-paramyosin ΔSal, to 95% completion in a total reaction volume of 10 μl in 5 hours at 4°C in 50 mM Tris-HCl (pH 7.5 @ 25°C) with 0.5 mM EDTA and 1 mM DTT. Storage Temperature: -20°C . Reaction Conditions 1X TEV Protease Reaction Buffer Incubate at 4°C 1X TEV Protease Reaction Buffer 50 mM Tris-HCl 0.5 mM EDTA 1 mM DTT (pH 7.5 @ 25°C) Storage Buffer 50 mM Tris-HCl 250 mM NaCl 1 mM TCEP 1 mM EDTA 50% Glycerol pH 7.5 @ 25°C Heat Inactivation 65°C for 10 minutes Molecular Weight Apparent: 28 kDa FAQ Q: Can I use Cold-Active TEV Protease (NEB #P8118) during the dialysis of my fusion protein? A: Yes. Cold-Active TEV Protease can be added to a sample before or during dialysis as long as the two buffers are compatible with Cold-Active TEV Protease activity. Q: Can Cold-Active TEV Protease (NEB #P8118) be used at temperatures higher than 4°C? A: Yes. Cold-Active TEV Protease can be used at temperatures between 4°C and 37°C. The incubation time of the reaction may be reduced when the incubation temperature is increased. Q: Can I inactivate Cold-Active TEV Protease (NEB #P8118) once my reaction is over? A: Yes. Cold-Active TEV Protease can be heat-inactivated by incubating the reaction at 65°C for 10 minutes. Q: Can Cold-Active TEV Protease (NEB #P8118) cleave a fusion protein bound to an affinity column? A: While we have successfully demonstrated that Cold-Active TEV Protease can cleave a tag from a tagged protein immobilized on an affinity resin, we recommend testing the cleavage reaction on the fusion protein eluted from the affinity column first to determine the best conditions. Q: Should I dialyze my reaction prior to cleavage with Cold-Active TEV Protease (NEB #P8118)? A: We recommend dialyzing the fusion protein if the buffer has > 2 M urea, > 1 M Guanidine hydrochloride, > 30% glycerol, pH values below 6 or above 9, or cysteine protease inhibitors. Q: What is the difference between Cold-Active TEV Protease (NEB #P8118) and TEV Protease (NEB #P8112)? A: Cold-Active TEV Protease is engineered for higher activity, especially at lower temperatures. Our data shows that Cold-Active TEV Protease is 3 times faster than TEV Protease at 4°C and at higher temperatures. Both enzymes can be used in the same 1X TEV Protease reaction buffer and can be inactivated at 65°C for 10 minutes.