Imidazole and Amicon concentrators

Bill Geese wjgeese at astro.ocis.temple.edu
Tue Dec 23 10:05:45 EST 1997


The protein is 32.6kD; the centricon cut-off is 10kD
I dont detect it in the filtrate nor in the retentate (Bradford and SDS 
PAGE); it's as if it is getting stuck to the membrane in some way and 
is unable to come off. Anyone else out there having prblems with centricon 
microconcentrators?

Thanks
BIll


On Thu, 18 Dec 1997, Lars Komorowski wrote:

> 
> Bill Geese schrieb in Nachricht ...
> >Hello all,
> >I am having problems concentrating my his-tag Ni-NTA purified protein
> >using Amicon's centricon-10 microconcentrator.  The specific problem
> >seems to be the concentration of imizadole in the elution of the target
> >protein from the Ni++ column.  THe fractions that I pool to
> >concentrate are in 60mM to 1M imidazole (final concentration).  After
> >passing these through the column as per instructions, I retain little to
> >no target protein (as determined by Bradford assay and SDS PAGE).
> >Recently I was informed by an Amicon technician that imidazole
> concentrations
> >higher than 300mM damage the membrane of the centricon column.
> >
> >Does anyone out there have any alternative procedures and/or product
> >recommendations that will work under high imidazole concentrations.
> >Diluting the sample to lower the effective imidazole concentration is an
> >option, however this bumps up the time to concentrate the protein
> >considerably.
> >
> >Thanks
> >Bill
> 
> Maybe you are experiencing problems due to the size of your protein. Have
> thought about the possibility of the protein passing through the membrane ?
> 
> 
> 
> 



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