Hi Nicola, 0.1-1mM Cysteine, GSH or BME will work fine. You can also try using very fresh TEV without reducing reagent (or store it with BME and remove the reducing reagent by some column just before use). Depending on how concentrated the stock is, diluting it 3-5x could also sufficiently reduce the redox potential of the solution. Another thing to try is to add some GS-SG to consume the DTT if it is already there. TEV is not a very stable protease due to both oxidation and aggregation. So yes, there is a strong reason to make it in the lab. We can usually make 20-40mg crude TEV from a liter of LB culture.
Zhijie > On Feb 13, 2019, at 1:47 PM, Nicola Evans > <0000251ca3b4615e-dmarc-requ...@jiscmail.ac.uk> wrote: > > I am interested in purifying proteins with 1 or 2 disulphide bonds, and have > been using enterokinase to cleave off N-terminal tags but we have had issues > with poor cleavage and want to try TEV cleavage instead. However TEV protease > is usually kept in a high amount of DTT and I am concerned about reducing the > disulphide bonds in my purified proteins. I have used TEV protease before > with 0.25mM TCEP and it worked well. Is there a way to use TEV with very > little or no reducing agent? Perhaps by optimising conditions such as adding > glycerol? We were thinking of buying in commercial TEV protease so any advice > on that is also welcomed (is there any merit, except cost, to making it > ourselves?) > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
