3 replies to this topic

[Miss_Delta_Notch]

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

[LifeTein Peptide]

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.


James
Understanding Life One Protein At a Time...
www.lifetein.com
A peptide Synthesis Company

[T C]

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

Best,
TC

Miss_Delta_Notch, on May 13 2009, 12:35 AM, said:

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

[waichun]

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.