Dear All
I am working on AlkB family proteins, Here I am facing a problem in protein purification step. I am using His-tagged protein and trapped and purified by Ni-resin column. I could purified the protein and it was shown from SDS-PAGE analysis, but problem is, it is precipitating after elution from column using 100 mM Imidazole-25mM K2HPO4 as a eluting buffer and it does not precipitating when I used pure 100mM Imd solution (bellow pH 7 and it is not suggested for protein activity assay studies.
can any one suggest me, what is the problem i am facing and how it can be solved, what are suitable condition and buffer should I use ?
after collection of protein, put it on dialysis to change the buffer.Use either phosphate buffer or PBS buffer with pH 7-8
Hi Srivardhan, Can you mention the pH of elution buffer? Have you tried reduced molarity of K2HPO4 or Na2HPO4?
Hi Srivardhan, You can adjust the same pH of pure 100mM Imd solution with pH of 100 mM Imidazole-25mM K2HPO4 .
Dear Hemant Kardam, Dear Hillol Chakdar, and Dear Sun Yan.
Thank you for your kind information. I have been following your suggestion, I will let u know if when I faced any problem.
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