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,