tags - what are you using? - his-tag, strep-tag, GST, MBP.... (Jul/30/2008 )

Hi there,

what kind of tags are you using to purify a specific protein?

Up to now I worked with His6-, Strep-tag, GST or MBP. What else could I use and which one could you highly recommend to me?

thanks in advance!

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hello I used His6- and GST.
i have a lot of problems about using GST tag. could you give me some help? many thanks to you! my MSN is kongjixiang@hotmail.com

stone

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We typically have His, GST or MBP as tags. I am currently developing a family of eight vectors based on the pETDuet vector family with both N- and C-tags for purification and/or solubility. His/FLAG, FLAG/His, T7/His, His/GFP, His/MBP, GST/His, Mistic/His and MBP/TrxA/His. We decided to go with MBP from Pyrococcus furiosus, as some papers suggested it solubilises "difficult" proteins better than other MBPs. Oh, yes, we work on membrane-bound complexes, which affects the tag selection.

Stone, what are your protocols for GST purification? We can't really help without knowing what you're currently doing...

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hi i use pEGX vector for GST fusion, and the purpose for the expression is to do the pull-down experiments. the protocol i use is Novagen company's GST-Bind kits.

i heard that it conformation of the GST after expressing is very important. So i am wondering that how can i make it the correct conformation?

thanks a lot

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I presume the protein is expressing in the soluble fraction, rather than as an inclusion body. What's the lysis buffer in the Novagen assay? What GSH beads are you using?

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the lysis buffer is Na2hpo4,KH2PO4,NaCL,KCL. PH7.3 Then Lysozyme, then sonication.

thanks

stone

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hello.I am doing protein purification with pGEX and i was wondering what were the different ways to cleave the purified product .I have transformed cells with prescision protease but does one follow the same steps to purify the protease after which do an on column purification of the my protein with the gst tag.

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If you look very very carefully in the Precission description, you might see somewhere that it's actually a GST-fusion protein itself. So when you cleave your fusion with Precission, you should be able to simply re-run the solution on a GSH column. The Precission and the GSt from the fusion protein should stick, and voila, pure recombinant protein (after you load onto your next column, concentrate, run on a gel filtration column... etc, etc, etc)

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thanks very much swanny, i would try. I think your suggestions help me a lot!

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