Rf: PM purification

LOGAND logand at msdos.ensam.inra.fr
Mon Jun 12 02:48:07 EST 1995


On 8 Jun 1995, Ken Shirasu wrote:

> Hello!  Netters!
>
> Does anyone knows good purification methods of plasma membrane from plants 
> (especially plant cell culture).  I would like to isolate plasma membrane 
> fraction from soybean cell cultures.     Thanks.
>
> Ken
>
>

>You should check out the Meth. Enzymol. volumes. 
>But here is what is done, in a nut shell:

>The tissue is ground up very gently: Earlier references talk about 
>mincing the tissue with a razor blade. I use a Waring blender to grind 
>4x10 sec. Buffer: 100mM HEPES, pH 7.5, 10%  Sucrose, 1mM EDTA, 1mM 
>MgCl2, and 1mM PMSF.

>The ground up tissue is filtered through several layers of cheese cloth.

>The filtrate is spun at 18,000xg for 20 min (twice).

>The resulting sup is spun at 100,000xg for 1h.

>The pellet is resuspended in grinding buffer.

>If this pellet (resuspended) which is composed of membranes, is 
>centrifuged through a sucrose density gradient, the plasma membrane 
>fraction will collect at 30/40 sucrose layers. One can check for the 
>authenticity of this fraction by assaying for a marker enzyme.

>H.S. Roychowdhury
>Plant Genetic Engineering Lab.
>Box 3GL, NM State Univ. 
>Las Cruces, NM 88003  Phone: (505) 646-5785                           
>hroychow at nmsu.edu                     


The sucrose isopycnic gradient method described above will give you an 
enriched fraction of PM relative to lighter endomembranes (ribosome 
free ER, tonoplast and Golgi (partially)) and the differential 
centrifugation removes the vast majority of whole plastids and 
mitochondria but does not give a very pure preparation at all. If you 
were to consider the above then a sucrose step suited to your material 
and smaller would be better ie. only a five percentage difference 
around the peak of PM marker enzyme activity as found on a linear 
sucrose gradient.

The method of choice is the aqueous-polymer two phase partioning method 
which is routinely used nowadays. The method may need some optimising 
depending on the plant material you are using (I'm sorry I can't give a 
complete protocol) ie. one  often has to change the dextran/PEG 
concentrations and the KCl concentration for an introduction see 
-

the original paper on this method:

        Separation of presumptive plasma membranes from mitochondria by 
        partition in an aqueous polymer two-phase system.
        Widell, S. and Larsson, C.
        Physiol. Plant. 51, 368-374. (1981)

also see the book-

The Plant Plasma membrane. Larsson and Moller (1990) Springer-Verlag.

For more recent papers I suggest a literature search and then checking 
the refs. in any paper using the technique to bring you closer to a 
working or starting method.

David C. Logan
INRA-ENSA(M)-CNRS
Montpellier
FRANCE

Logand at msdos.ensam.inra.fr





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