Have you tried expression tricks like Rosetta cells? Testing different colonies and/or starting from fresh transformants? Sometimes that matters.
If your protein is an oligomer and your contaminants are degradation products, you might try adding some urea. If desparate, you could spike the fraction collector tubes with EDTA when you run the Ni column, as well as using the usual protease inhibitors. Phoebe ===================================== Phoebe A. Rice Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp ---- Original message ---- >Date: Thu, 26 Aug 2010 12:07:45 -0400 >From: CCP4 bulletin board <[email protected]> (on behalf of Matthew >Bratkowski <[email protected]>) >Subject: Re: [ccp4bb] Problems in purification >To: [email protected] > > 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 > <[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
