Separation of inclusion bodies from cell debris - (Jan/27/2008 )
I want to separate inclusion bodies from E.coli from the cellular debris after lysis of the cells.
Can I do this by normal centrifugation? and if yes, at what speed?
I mean first I would use a "low speed" centrifugation to get rid of the debris and the inclusion bodies should stay in suspension and in the second higher speed step, i like to spin down the inclusion bodies.
Is that possible and and which speeds (rcf)?
You could spin the cell debris at 500 -1000x g and get them down. Then you could spin at a 100,000x g for everything else.
I would suggest that you do 500, 6000, 14000, 100,000 x g to know which fractions the inclusion bodies do come down. You could alter the g's according to your needs.
people do sub-cellular fractionation in this method. They also do a density gradient centrifugation (like sucrose or ficoll) to clean the samples.
I also purified inclusion bodies in relatively low speed centrifugation. The important thing is washing the pellet
(inclusion bodies) before moving on.
First i wash the cell pellet with Native lysis buffer for 60 min to get rid of all the soluble proteins (10,000xg, 30min)
than i wash twice the pellet with 1XPBS+1% TRITON-100 (very important) at 30,000xg, 30 min. then use denaturing
lysis buffer- 8M UREA for 60 min (most of the pellet is not soluble) and centrifuge at 38000xg, for 20min.