Rf: Subcellular Fractionation

LOGAND logand at msdos.montpellier.inra.fr
Mon Nov 14 12:09:27 EST 1994

Kieran Breen wrote:

>We are studying the cellular expression of an enzyme which can exist both
>in the Golgi and at the level of the cell membrane. In vivo, subcellular
>fractionation is relatively straightforward using a differential gradient
>However, I have not come accross a parallel method for separating the
>plasma membranes and Golgi fractions from a cultured cell monolayer. This
>is a particular problem when the small amount of starting material is taken
>into consideration. Has anybody elso come accross this problem, or how it
>may be addressed?

I assume that the same methodology can be used and that the membranes will have 
the same equilibrium densities and thus the problem is only one of amount of 
material. Can you not just run smaller gradients? For instance if you have 
access to a Beckman TL-100 benchtop ultra and the very small swing out rotor 
(TLS-55 I think) or similar, then you can run gradients of less than 1ml (step 
gradients and linear gradients using the freeze thaw method) and this might 
solve your problem.


Logand at msdos.montpellier.inra.fr

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