questions about centrifugation and organelle lysis - (Sep/28/2012 )
I have questions. I`m using Percoll gradient centrifugation to get endosomes and lysosomes ( for studying the intracellular fate of labled macromolecule). Could anybody give some suggestions how to lyse vesicules after I have collected the fractions?(Can I use Triton 100X 0.1-0.5% like on cells? And how could I separate whole cells from nuclei in pellet, how should I centrifuge?(if its even possible) (from HeLa cells)?
The nuclear fractionation step is usually the first step in these sorts of protocols. It usually takes some optimisation to get it right, but it should work OK if you use a potter-elvehiem homogenizer (ball head, not cylindrical) with the correct clearance. Nature Protocols has a very good protocol for doing this, but I can't find my copy at the moment.