Centrifugation (View forum version)



TomH

Posted 27 May 2010 - 04:14 AM

Hi all,

I am spinning a protein sample at 100000g for 1 hour to determine if there are any aggregates in the solution. My question is how can I work out what range of molecule sizes I'd expect to sediment under these conditions? I know there is some formula somewhere that would tell me, for example, at that speed and time I'd expect anything over X kDa to sediment. Does anyone know how I can work this out?

Inmost sun

Posted 27 May 2010 - 06:24 AM

Hi all,

I am spinning a protein sample at 100000g for 1 hour to determine if there are any aggregates in the solution. My question is how can I work out what range of molecule sizes I'd expect to sediment under these conditions? I know there is some formula somewhere that would tell me, for example, at that speed and time I'd expect anything over X kDa to sediment. Does anyone know how I can work this out?


you mean to determine a sedimentation coefficient? You should use a sucrose density ultracentrifugation utilizing reference proteins with known molecular mass AND sedimentation coefficient;

follow the protocol of Martin & Ames (1961), JBC 236, p 1372-1379

mdfenko

Posted 28 May 2010 - 10:35 AM

once you know the sedimentation coefficient then you can calculate "clearing time" (the formula is or was given in a book that comes or came with beckman ultracentrifuges. the beckman coulter website has some calculators.

then clearing time (in hours), t=k/s. s=sedimentation coefficient and k= a speed factor that you can calculate for your rotor (at a given speed or rcf) with the calculator at the website.