This short paper in Nature Methods is probably worth a read and may help with your Ni-NTA issues:
http://www.nature.com/nmeth/journal/v6/n7/full/nmeth0709-477.html Nature Methods 6, 477 - 478 (2009) doi:10.1038/nmeth0709-477 Enabling IMAC purification of low abundance recombinant proteins from E. coli lysates In short, they detail that removing E. coli periplasm prior to cell lysis, or spiking lysates with 2 mM FeSO4, may improve purification of low abundance proteins on IMAC resins - their theory being that removal or saturation of metallphores (metal chelators) will prevent Ni2+ leaching from the IMAC resin. If possible avoid using an NaCl concentration as low as 20 mM, as there will be lots of non-specific interactions at such a low ionic strength. Roger On 26 August 2010 13:24, ganesh pathare <[email protected]> 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 -- Roger B. Dodd Cambridge Institute for Medical Research Wellcome Trust/MRC Building Hills Road Cambridge CB2 0XY UK
