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6his-tagged protein purification

6his-tagged protein purification - Protein Science

6his-tagged protein purification - Post Questions and Discuss Protein Structure and Function, Protein Localization etc.. Please do not post Proteomic questions here, there is a Proteomic Forum below.


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Old 02-18-2007, 06:43 PM
Pipette Filler
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Default 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??

thanks

Last edited by proteoglycan; 02-18-2007 at 08:14 PM.
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Old 02-18-2007, 08:49 PM
Pipette Filler
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Default Re: 6his-tagged protein purification

Quote:
Originally Posted by proteoglycan View Post
I tried dialysis and lost approx. 3/4 of my starting protein concentration (after purification).
Hi! As I truly understand firstly you made purification under denaturating conditions in 6M urea. So afer purification you made dialysis and lost your protein. So what Mw of your protein and Mw cuttoff of your membrane? The really Mw of denaturated proteins are smaller then native. To avoid loss of your protein use Dialysis tube with smaller cutoff/ For ex when i dialysed my protein with Mw 24kDa I used DT cutoff 8kDa. If I used DT cutoff 12kDa I lose my protein

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
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