I have utilized pET26b(+) NS2B-NS3 DENV2, and transfected it in E.coli. After transfection the selected cells were characterized based on the kanamycin resistance present in the vector. The selected cells were incubated with 1 millimolar of IPTG at 28 C for 4 hrs induction. While the results seem positive for the experiment, multiple bands occur underneath the protein of interest. the selection was done based on the histidine tag using penta-his antibody. Please help me with this concern. I am using TMB for the development of the blot.
I think it likely that these are degradation products of the full length protein (assuming you loaded the same amount of lysate or protein in the non-induced as the induced lane).
Dear Dr. Michael J. Benedik ,
Thank you very much for your reply.
I have to decrease these degradation products as I require the highest purity of this protein (enzyme), to set enzymatic assay in the upcoming experiment, and then the study will go in vivo, please help me with your suggestions.
Best
Mohit
If you have not yet tried adding protease inhibitors when preparing the protein lysate then that would be the first thing I would try. Secondly you might reduce the time after induction or reduce the temperature a bit more.
Lastly the level of these products is fairly minor and you might just ignore them.
However I would note that you DO NOT know if your protein is pure or not. Since you are doing a western blot by definition you are only seeing the proteins that are detected. You might wish to run a protein gel and stain to get an idea of the actual purity, if this is something important to you. But truthfully for many applications you do not need pure protein.
Dear Dr. Michael J. Benedik
Thank you very much for your prompt reply and suggestions, I will do as suggested.
Sincerely
Mohit,
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