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I assume you are trying to do a non-denaturing prep. Have you run your sample on a size exclusion column to see if it is aggregated? If it is aggregated, it can stick to a lot of contaminating proteins which will be difficult if not impossible to separate. ho > On Thu, Aug 26, 2010 at 8:24 AM, ganesh pathare <[log in to unmask]>wrote: > >> 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 >>