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" <jubo...@jhsph.edu> Para: CCP4BB@JISCMAIL.AC.UK 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" <rashmi.panigrah...@gmail.com> >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/
