Hello everyone!,
I would like to know if someone can help us with this issue.
We are using TEV PROTEASE in my Lab following the manufacturer instructions (Genscript, USA) and we have had bad results for our protein. We have reviewed the protocol and the cut signal sequence and we think that all is correct.
I would like to know if someone is using this protease and if someone could give me some advice or protocol to do the process.
Thank you very much in advance.
Best wishes,
Marcos.
Dear Marcos
first of all, i suggest to produce the recombinant Tev by your self using the plasmid of David Waugh shared by Addgene (https://www.addgene.org/8827/).
You can easly produce mgs if Tev and save a lot of money.
Attached you can find some information about tev.
i never used the genscript tev but i used actev of thermo in the past amd actually the ones handmade and they work similarly. Generally i perform digestion O/N at room temperature in tris 20mM, edta 0.5mM and dtt 1mM ph=8 buffer and i'm using at least 2ul of Tev for 1mg of protein and protein concentration >= 1mg/ml to maintain also tev concentration at least 2-5ug/ml.
did you test your Protocol in just one protein or did you obtain negative results for several protein? because some time ( if your protein is aggregate for example) the absence of cutting is due to the protein, therefore to be sure that the problem is related to the tev or its protocoll could be usefull to perform also digestion of a positive control that you know that have to work.
good luck
Manuele
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