Imidazole and Amicon concentrators
Bill Geese
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
Lars Komorowski
Bill Geese schrieb in Nachricht ...
Maybe you are experiencing problems due to the size of your protein. Have
thought about the possibility of the protein passing through the membrane ?
Bill Geese
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
Randall C Willis
Bill Geese wrote:
>
> 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.
Bill,
We've had innumerable similar experiences and it is due to the fact that
your protein may indeed be stuck to the membrane...to verify, scrape a
piece of the membrane off into a sample of SDS loading buffer and boil
the hell out of it for a while. If it is stuck, Millipore and
Pall/Gelman also make concentrators which are supposedly lower binders
but, in our experiences, the Millipore product can be imperfect as
well. I would suggest that you contact reps for each company and ask
for a sample of their concentrators.
Good luck,
Randall C Willis, Researcher
Biochemistry Research, Hospital for Sick Children
3522-555 University Ave
Toronto, ON
M5G 1X8 CANADA
416-813-5933 (ph) -5022 (fax)
wil...@gandalf.psf.sickkids.on.ca
Randall C Willis, Publisher
Aliquotes Press
Aliquotes: A Journal Of Molecular and Biochemical
Humour and Information
58 Balfour Ave.
Toronto, ON
M4C 1T6 CANADA
416-691-2921 (ph) 416-813-5022 (fax)
ro...@xybercom.net
Peter Ashton
Bill Geese wrote in message ...
<SNIP>
>> >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
You could try a desalting column to get rid of the imidazole...That
shouldn't have too
drastic effect on your protein concentration, but this could be a problem if
your sample
size is too big. A PD-10 column works up to 2.5 ml, five HiTrap desalting
columns
screwed together should give you 7.5ml, and both will give more if you only
want to
reduce the imidazole concentration rather than eliminating it completely.
Since you have quite a large protein you could also try dialysis...it still
works even
though some people seem to try to avoid it these days. And, of course,
dialysing
against a high salt buffer will concentrate for you at the same time...isn't
osmosis
wonderful?
Peter Ashton
The University of York, Department of Biology
nospam:pd...@york.ac.uk
Wolfgang Schechinger
How do you measure protein concentration?
On 27.12.18 03:01, m.golg...@gmail.com wrote:
> Hi, I'm in cas9 purification with the help of ni-nta agarose I use amicone 100 for further purification After using the amicone, my protein concentration decreases I do not know why
> I use 500 mg of imidazole for purification Please guide me Thank you
> _______________________________________________
> Methods mailing list
> Met...@net.bio.net
> http://www.bio.net/biomail/listinfo/methods
Dr Engelbert Buxbaum
huba...@gmx.de says...
in addition, it is possible that the protein precipitates as the
concentration increases. It may even accumulate on the membrane.
--
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