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GST tag fusion protein--- help please

GST activity

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4 replies to this topic

#1 sophia613

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Posted 14 July 2013 - 09:58 PM

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 ?

#2 Ahrenhase

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Posted 16 July 2013 - 04:46 PM

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.

#3 sophia613

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Posted 17 July 2013 - 03:50 AM

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!

#4 Missle

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Posted 17 July 2013 - 04:29 AM

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

#5 sophia613

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Posted 17 July 2013 - 05:31 PM

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?




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