purification and binding of protein in denaturing conditions - (Jan/18/2013 )

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

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

Do you changed your buffer with the binding buffer instead of 8M Urea, can you detail on how you change your buffer?