Hi
I need suggestion about my current problem. I purified His-tagged protein using Ni-NTA resin and enough amount and purity of protein was observed on SDS-PAGE. But the protein was precipitated soon after purification. I used 50 mM Tris pH8, 500 mM NaCl, 20 mM imidazole buffer for sonication of E. Coli, Ni-NTA binding and washing of resin. Then protein was eluted by 50 mM Tris pH8, 500 mM NaCl, 500 mM imidazole. I think this is caused by buffer pH because estimated isoelectric point of the tagged protein is pH8.2 - 8.4. I need to try lower or higher pH.
I do need your idea and suggestion.
Start by doing that, lower the pH. If that is not enough you can try diluting the sample and/or adding glycerol or some other stabilizing molecule.
The precipitation of your protein is possibly caused by the pH, try to lower your pH, maybe you can try to purify your protein in 20 mM PBS, 500 mM NaCl, pH 6.5. If there is no improvement, then maybe the region of your protein is not proper, you can try to extend the region.
I'd recommend eluting your protein from the column with a shallow gradient (or a step gradient) to your 500mM imidazole buffer. This will a) keep your protein concentration lower which will help prevent precipitation and b) will keep the imidazole concentration in your protein solution lower (high concentrations of imidazole can destabilize proteins...or so I've heard). You may also want to consider adding EDTA to your fractions in case the Ni2+ that is stripped from the column upon elution is "bad" for you protein.
Hi All
I followed the advice of Omar Gonzalez-Ortega and Jiuyang Liu, I used 20 mM phosphate buffer pH6.5 and pH7, both contains 150 mM NaCl, 20 mM Imidazole for sonication, protein binding to Ni-NTA and washing the resin. And eluted the protein considering James Nicholas Vranish's advice using lower imidazole (20 mM phosphate pH6.5 and pH7 containing 150 mM NaCl, 100 mM Imidazole). The eluted protein could be concentrated to at least 0.5 mg/ml. So the result was good and the pH6.5 looks like better than pH7.
I appreciate you.
If you have any other idea, please give me.
hello, you can use a Tris buffer of pH 7, add upto 10 percent glycerol and 2 mM bMe in your lysis and wash buffer, after elution you may add some more bMe upto 5 mM in case it prevents aggregation
- Badges
- Science method