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GST- protein, batch purification


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#1 Miss_Delta_Notch

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Posted 12 May 2009 - 10:05 AM

hi,
i've tried to purify my GST- fusionprotein by Glutathion Sepharose medium. westernanalysis showed, that theres nearly no fusionprotein after elution with glutathione. by analyzing the flow- trough of the fist two washing steps, it comes clear that a lot of the GST- protein is actually in the flow- trough and doen'T bind to the beads. Besides, theres a lot of degradation of the fusionprotein. this can be prevent by using more inhibitors. does anybody know why the GST- tagged protein doen'T bind to the beads?
thanks :D

#2 LifeTein Peptide

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Posted 12 May 2009 - 05:33 PM

It is better to dialyze your GST protein first and then purify. GST is a really easy protein to work on. Keep your process in the cold room and add protease inhibitors. The cocktail tablet from Roche is good.


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#3 T C

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Posted 12 May 2009 - 09:31 PM

You can also try DNA precipitation...that helps and it improves binding.

Best,
TC

hi,
i've tried to purify my GST- fusionprotein by Glutathion Sepharose medium. westernanalysis showed, that theres nearly no fusionprotein after elution with glutathione. by analyzing the flow- trough of the fist two washing steps, it comes clear that a lot of the GST- protein is actually in the flow- trough and doen'T bind to the beads. Besides, theres a lot of degradation of the fusionprotein. this can be prevent by using more inhibitors. does anybody know why the GST- tagged protein doen'T bind to the beads?
thanks <_<



#4 waichun

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Posted 19 May 2009 - 05:37 AM

did you play around with the parameter. eg volume of GST beads used, suggested by the protocol?
i had the same problem as yours before but when I increase the volume of GST beads for binding it became alright.
At every protocol about GST protein purification there is always one sentence: optimal condition varies from proteins to proteins and you have to find it empirically. It's painful and takes time but also very true.




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