Dear All,
I am new in molecular biology and biochemistry and trying to purify one of the protein for which I have done cloning and confirmed it by sequencing. Cloned vector has his-tag and its expression profile shows no solubility. So, I try purification under denaturing conditions using 8M urea by re-suspending the pellet. And in denaturing condition, I believe that protein is in open conformation and should bind to Ni-NTA beads. But in my case most of the protein goes in flow thru, which may be hard to believe.
Please suggest how to increase binding of protein to Ni beads to do purification.
Also I want to ask if anyone has used 1M NaCl to increase binding in such cases as I have read at many places that 1M NaCl helps in binding of protein. But I dont knw how NaCl will behave in the presence of 8M urea. Any help will be highly appreciable.
Thanks and Regards
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purification and binding of protein in denaturing conditions
Started by shivu11, Jan 18 2013 11:00 AM
poor solubility protein purification poor solubility protein purification
2 replies to this topic
#2
shivu11
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Posted 18 January 2013 - 11:00 AM
Dear All,
I am new in molecular biology and biochemistry and trying to purify one of the protein for which I have done cloning and confirmed it by sequencing. Cloned vector has his-tag and its expression profile shows no solubility. So, I try purification under denaturing conditions using 8M urea by re-suspending the pellet. And in denaturing condition, I believe that protein is in open conformation and should bind to Ni-NTA beads. But in my case most of the protein goes in flow thru, which may be hard to believe.
Please suggest how to increase binding of protein to Ni beads to do purification.
Also I want to ask if anyone has used 1M NaCl to increase binding in such cases as I have read at many places that 1M NaCl helps in binding of protein. But I dont knw how NaCl will behave in the presence of 8M urea. Any help will be highly appreciable.
Thanks and Regards
I am new in molecular biology and biochemistry and trying to purify one of the protein for which I have done cloning and confirmed it by sequencing. Cloned vector has his-tag and its expression profile shows no solubility. So, I try purification under denaturing conditions using 8M urea by re-suspending the pellet. And in denaturing condition, I believe that protein is in open conformation and should bind to Ni-NTA beads. But in my case most of the protein goes in flow thru, which may be hard to believe.
Please suggest how to increase binding of protein to Ni beads to do purification.
Also I want to ask if anyone has used 1M NaCl to increase binding in such cases as I have read at many places that 1M NaCl helps in binding of protein. But I dont knw how NaCl will behave in the presence of 8M urea. Any help will be highly appreciable.
Thanks and Regards
#3
Adrian K
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Posted 20 January 2013 - 08:56 AM
Do you changed your buffer with the binding buffer instead of 8M Urea, can you detail on how you change your buffer?
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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
