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Purifying recombinant protein from pellet?


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#1 Scienceboy

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Posted 04 April 2012 - 03:51 AM

Hi

I am producing a protein in E.coli and the problem is that it is not soluble. For purification I am using GE healthcare spin trap for purifying the His tagged protein (http://www.gelifesci...moduleid=166507).

I decided to test if you can purify the protein from the pellet so I just resuspended the pellet to 0,5 ml buffer and just purified the protein using the kit and got nice results on SDS-page.

Can this protein be functional?

Cheers!

#2 Laforet

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Posted 05 April 2012 - 01:21 PM

Depends on the protein however most insoluble proteins are not properly folded and hence unlikely tO be active

#3 protolder

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Posted 09 April 2012 - 03:01 PM

Hola, I guess that the trucks for express your protein soluble, failed. It could occurs that the used buffer from the kit, gives your protein free, broken any interaction with other insoluble protein and your protein was functional, but could occurs that your protein forms inclusion bodies and you are purificating them in the resin. Two things to help you to distinguish it: soluble proteins elutes about 0.2mM Imidazole and aggregates about 0.4 or more. / 2.- You could load a well i a SDS gel electrophoresis with loading buffer without reducer and withouth boil the sample. If your protein continue migrating in the correct position could be functional but if you see multimers and in the natural form they don´t exist probably you need to denature and refold with the risks that it carries. Buena suerte




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