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Rashmi,
 
I had a similar problem when I used an amicon to concentrate my protein, your buffer composition indicates me that a lot of salt concentration and glicerol makes your protein soluble, I had a similar buffer with a lot of saltt and 15% glicerol, and I simply reason that my protein it is actually soluble but Temperature was also critical so I decided to take the centrifuge inside of a refrigerator and I centrifuge at 10°C or less and my protein survive the process of a possible thermal shock that the protein is induced, sometimes something simple works fine and it did for me.
 
hope this help you.
 
aaron hernandez
Universidad nacional autónoma de Mexico UNAM
 

De: "Bosch, Juergen" <[log in to unmask]>
Para: [log in to unmask]
Enviado: Domingo, 29 de enero, 2012 11:08:09
Asunto: Re: [ccp4bb] protein lost on membrane of centricon!!

Another option I didn't mention is presoak your centricons in 30% glycerol over night prior to usage.
Jürgen

On Jan 28, 2012, at 12:47 PM, Bosch, Juergen wrote:

Are you close to the theoretical isoelectric point of your protein ? Change pH of buffer
Jürgen

......................
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:      +1-410-614-4894
Fax:      +1-410-955-3655
http://web.mac.com/bosch_lab/

On Jan 28, 2012, at 10:55, "rashmi panigrahi" <[log in to unmask]> wrote:

Hi all,
I tried to concentrate my protein using vivaspin 20  10,000 MWCO PES.
The protein was in 50mM Hepes pH 7.5, 500mM KCl  and 10% glycerol.
I lost about 90% of my protein on the membrane of the centricon.

Please  suggest some way of concentrating this protein.
Will concentrating using peg 20K be a good alternative??

regards

rashmi

......................
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:      +1-410-614-4894
Fax:      +1-410-955-2926
http://web.mac.com/bosch_lab/