Imidazole and Amicon concentrators
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
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
> >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
> 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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