Dear all, I have problems in purifying a protein. The protein is 38,000 daltons and has a N-ter His-Tag. The protein expression levels are low and as a result I have a limit for the purification steps. Initially I used NiNTA columns with 50 mM sodium phosphate buffer pH8, 300 mM NaCl, 20 to 250 mM Immidazole for the affinity purification, but it contains lot of impurities. I varied the salt concentrations out of which I could get optimal results at 20 mM NaCl concentration but still the amount of impurities was more. After affinity purifications I used Ion exchange chromatography using MonoQ column (25 mM tris pH 7.5, NaCl 0 to 1M) which could not seperate the protein from the impurities. I also tried using Hydrophobic interaction chromatography (Resource Ether, Phenyl sepharose, Resource Isopropyl) instead of ionexchange chromatography, which resulted in better purification of the protein, but the problem is I get very less protein after this step and there are still two major impurities. The buffer conditions for HIC was (1.5 M ammonium sulphate, 25 mM Phosphate buffer pH 7).
I would be very greatful if someone could help me in this concern. Thanks in advance. Regards, Ganesh
