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Protein Purification by precipitation


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#1 biotef

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Posted 26 August 2010 - 10:09 PM

my protein has a pI of approx 5.6 - 6.0 and we maintain pH of 4.5. our protein is produced in Ibs and we solubilise refold it and concentrate it using TFF. on concentrating the protein solution shows some high molecular weight contaminants [observed in sds page] in addition to our protein. is there any percipitation or simple way to get rid of these contaminants. we then perform ion exchange chromato at pH 4.5 but these contaminants still persist. and our volume after IEX is too large to laod to GPC. AnNy suggestion is welcome.

thanks.

#2 protolder

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Posted 26 August 2010 - 10:20 PM

Hola Firstly I thoudht that the upper bands were multimer, but you use denaturing PAGE,and I think this is the clue. Probably if you run a well withouth reducer it would appears a hihger band of your protein and the contaminant. So you have the contamination specifically bounded by disulphure bridge to your protein, wich you broke in PAGE. So add a bit of reducer in the tangential filtration or in the ion exchage buffer. If you put it I would increase it. Good luck

#3 biotef

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Posted 27 August 2010 - 12:21 AM

Hola Firstly I thoudht that the upper bands were multimer, but you use denaturing PAGE,and I think this is the clue. Probably if you run a well withouth reducer it would appears a hihger band of your protein and the contaminant. So you have the contamination specifically bounded by disulphure bridge to your protein, wich you broke in PAGE. So add a bit of reducer in the tangential filtration or in the ion exchage buffer. If you put it I would increase it. Good luck

Actually we perfrom both reducing and non reducing page and these are not multimers. yes to some extent the intensity of some band decreases but we get number of bands and we are unable to get rid of them.




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