shivu11

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 trying to clone a 900bp gene. I have amplified the gene from genomic dna. and i have added NDE1 and XHO1 sites. I have been trying to clone this gene since 6 months, but no success. I do digestion for 3hrs then gel extract it and do ligation for 16hrs at 15degree. Then transform it in DH5a, I get nice colonies after that. I isolate plasmid from 2-3 colonies and do RE digestion and PCR for confirmation. In PCR I get number of bands, inculding one at 900 and others at 3000, 2000bp. and when I do digestion I get two bands, one at 5000( vector) and one between 2500 and 3000bp. I dont knw what is happening. Please help. I have to clone this gene.
Thanks in advance.

Hi All,

i have cloned one of the bacterial protein in Pet28c vector with a His-tag at N-terminal which i have confirmed by sequencing.

I have tried overexpressing it using IPTG. and it gives excellent expression and solubilty at 37, 25 and 18 degrees. But when i try purify it using Ni-NTA coloumn, all the protein comes in flowthru and further washing with 40mM immidazole with other impurities and no protein in elution at 250mM immidazole. I have added 50mM tris buffer with 50mM NaCl and 5% glycerol.

Can anyone please suggest what could be done to increase the binding of the protein with coloumn.

thanks in advance,