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.
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Recombinant protein from cell lysate
Started by proteinsolutions, Aug 29 2011 09:42 AM
sephadex column lectin column 130KDa protein
4 replies to this topic
#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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..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#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
Adrian K, on 29 August 2011 - 10:34 AM, said:
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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