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Recombinant protein from cell lysate

sephadex column lectin column 130KDa protein

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#1 proteinsolutions

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Posted 29 August 2011 - 09:42 AM

Hi, I am trying to purify a recombinant unlabelled glycoprotein protein from a mammalian cell lysate. Can any expert suggest, which are the shortest ways to purify this protein? I am using lectin affinity followed by sephadex 200 for purifying this 130 KDa protein. I tried 2 times i get it on SDS page but no immunoblot activity. I suspect protein gets denatured.
I need some suggestions.

#2 Adrian K

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Posted 29 August 2011 - 10:34 AM

did your unpurified crude extract shows immunoblot activity???
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#3 mdfenko

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Posted 29 August 2011 - 10:54 AM

the protein is denatured by sds-page. you need to use an antibody qualified for immunoblotting.
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#4 proteinsolutions

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Posted 31 August 2011 - 04:13 AM

did your unpurified crude extract shows immunoblot activity???


Yes, It blots with monoclonal, even the elute fraction blots but the immunoactivity is much decreased. Also the undesired protein bands sustains in the purified fraction.

#5 proteinsolutions

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Posted 31 August 2011 - 04:14 AM


Yes, It blots with monoclonal, even the elute fraction blots but the immunoactivity is much decreased. Also the undesired protein bands sustains in the purified fraction.

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