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6his-tagged protein purification
I have successfuly purified my 6his-tagged protein using a Ni-agarose column. However the 250mM Imidazole elution buffer may interfere with my spec assays that are done at 400nm wavelength. I did do some reading and imidazole absorbs light around 220nm. However, I would feel more confident with my results if elution buffer was out of the picture. I tried dialysis and lost approx. 3/4 of my starting protein concentration (after purification). I would like to try pH elution without denaturing conditions. My protein elutes around pH 4.5. The elution buffer I currently use contains 50mM NaHPO4, 300mM NaCl, and 250mM Imidazole pH 7. Can I use a volatile agent ie. acetic acid to create a pH gradient in the existing buffer excluding imidazole??
Last edited by proteoglycan; 02-18-2007 at 07:14 PM.
Re: 6his-tagged protein purification
If your add acetic acid to buffer of your composition I think you do not make it volatile Because as i know only ammonium acetate is volatile! I don't understand your strategy if your use spec measurment at 400nM
|6histagged , protein , purification|
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