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GST tag fusion protein--- help please - (Jul/14/2013 )

Hey there,

I've been having a hard time to purify a GST tag protein. I use the pGEX4T-1 vector and so my protein is GST tagged.The fusion protein is partialy soluble.Then I purified my protein ,but there was no activity.Is there any one can help me ?

-sophia613-

Is the protein at the approriate MW on a western? Perhaps the GST fusion is impeding enzymatic activity? If this is the case, there are several cleavable linkers you could use.

-Ahrenhase-

Thanks,Ahrenhase

I`ve purified the fusion protein by the Glutathione Sepharose ,the sds-page results is right .Now I am tring to cleavage the GST tag by THROMBIN.Wish me good luck!Thank you again!

-sophia613-

Good Luck! GST often dimerizes taking your target protein with it. This can interfere with functionality. Cleaving seems like a great idea.

-Missle-

Thank you,Missse
I have tried several times to purify the protein,and I it`s really a hard work .Could you give me some advise about using thrombin to cleavage the FUSION?

-sophia613-