Hi. What size are the impurities? If they are smaller than your protein, then they could actually be truncation products, which will be difficult to purify away since they maintain some of the same characteristics as the full length protein. You can check for C-terminal truncations using a His-antibody, but N-terminal ones will be harder to detect. If the impurities are larger (particularly if they are around 70 kDa), you could be looking at E. coli chaperones.
To improve, the purity of the first Ni-NTA step, I would include a more stringent wash. How many column volumes do you wash with now, and how high of imidazole concentration? You can go up to 20 mM Imidazole in your wash. You could also include some glycerol in your buffer (up to 10%) and betamercaptoethanol (around 5 mM) to break non-specific protein interactions. For ion exchange, run a shallow gradient and include more column volumes of wash before elution. For the third step purification, I would recommend using size exclusion chromatography. Either Superdex 200 or Sephacryl S-100 would probably work to remove some impurities as long as the impurities are a different size than your protein of interest. I would use between 150 mM - 1 M NaCl in the buffer, depending on how strong the non-specific interaction is, and 1 - 2 mM DTT. Make sure to collect small fractions (0.3 - 1.5 mL) to reduce contamination from nearby peaks. Matt On Thu, Aug 26, 2010 at 8:24 AM, ganesh pathare <ganeshpath...@gmail.com>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 >
