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heme protein precipitation

heme precipitation

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4 replies to this topic

#1 Biogareth

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Posted 24 April 2014 - 08:38 PM

Hello all,

 

Recently, we attempted to purify his-tag heme protein by Ni-NTA resin. Everything goes well, and we got elution fractions like wine. Elution buffer contains 250 mM imidazole, 50 mM tris-Hcl (pH 7.5) and 400 mM NaCl. But the protein started to precipitate when it came to the dialysis step that aims to remove imidazole. After the overnight dialysis at 4 degree, proteins with red color almost precipitated.     It seems that imidazole is essential for the enzyme stability. Do you think is it possible? Any suggestions are welcome!

Thanks!

 

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#2 mdfenko

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Posted 25 April 2014 - 03:55 AM

what is the composition of the dialysis buffer?

 

is the pH of the elution buffer adjusted before or after addition of imidazole?

 

the relatively high salt concentration in the dialysis may be causing the precipitation.


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#3 Biogareth

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Posted 25 April 2014 - 08:44 AM

The dialysis buffer is 400 mM NaCl and 50 mM Tris-HCl (pH7.5). We do find the pH increasing to 8.5 in the presence of 250 mM imidazole.



#4 mdfenko

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Posted 28 April 2014 - 04:14 AM

you might want to try raising the pH of the dialysis buffer to around 8.5 and see if it stays in solution.


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#5 labtastic

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Posted 06 May 2014 - 08:44 AM

Imidazole rings can bind heme groups (depending on the protein), so it might actually be stabilizing your protein. Perhaps just dialyze into low imidizaole (10-20 mM) rather than getting rid of it completely. After all, it had to be soluble in low imidazole concentrations for it to originally bind the resin.






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