What this indicates is that your protein is not in 'real inclusion bodies' but is merely precipitating - it's not even necessarily true that it's a precipitate inside the cell - instead it could be falling out of solution as you're lysing and so forth. It also may be binding to nucleic acids and then slowly coming off as NA get hydrolyzed or physically broken down.
Artem _____ From: megha goyal [mailto:mgbio...@gmail.com] Sent: Friday, October 30, 2009 6:32 AM To: Artem Evdokimov Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] Inclusion bodies centrifugation Tried out the method, but our protein is also lost in the supernatant. i collected the supernatant after 1st centrifugation and centrifuged it to obtain the pellet. we observed that our protein is present in that pellet and in further washes also we see protein loss too. Thanks anyways for all the suggestions. On 10/27/09, Artem Evdokimov <ar...@xtals.org> wrote: IB's precipitate in a few minutes at max. speed of tabletop Eppendorf. Almost any speed above 5-6K in a larger rotor will precipitate them in 10-20 minutes, provided that the density of your IB wash is normal (i.e. you're not using 70% sucrose, Renografin, or Cesium Chloride etc). In other terms, no worries. You have the technology. Artem _____ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of megha goyal Sent: Tuesday, October 27, 2009 10:21 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Inclusion bodies centrifugation Dear All, Our protein is expressed as inclusion bodies and I want to separate inclusion bodies from E.coli from the cellular debris after lysis of the cells by sonication. Can I do this by normal centrifugation? and if yes, at what speed? Our centrifuge has maximum speed of 14000 rpm. Can we do the separation using this centrifuge and if so how. Thanking in anticipation.
