SUBJECT: Isolation of plant proteins
DATE: 10/95


I'm trying to develop a protocol for the isolation of plant proteins that
would be suitable for a Freshman level lab course. I have found a variety of
sophisticated approaches, but I feel that these may be too difficult for the
students. I'm looking for a reliable procedure for breaking open cells from
roots, stems, and leaves in order to extract most or all of the cellular
proteins. These would be examined using SDS-PAGE.

My experience is with animal cells and I'm not familiar with any problems
there might be in dealing with cell walls when disrupting plant cells.

Any general-use procedure or standard reference(s) would be greatly appreciated.

Many thanks.

George

George Edick
RPI - Dept. Biology
Troy, NY 12180
edickg@rpi.edu


An easy way is to grind the tissue with a pestle and mortar directly in the
SDS/buffer solution you use for the samples to be loaded onto the gels.
Small volumes can be boiled and then centrifuged in microfuge/eppendorf
tubes. Alternatively- if you are allowed to use this with students- grind
the fresh tissue with a little liquid nitrogen in the pestle and mortar
before adding the extraction buffer- this helps to get a fine powder of
tissue before extracting into buffer. Hope this is of use. I assume that
you are only interested in running the samples on gels and not attempting
to measure enzyme activities?

From: at6@st-andrews.ac.uk (Alyson Tobin)


Have you tried using lyophilized plant tissue and then grinding it in an
SDS solution? I know that it works well when extracting plant DNA, but I
haven't tried it with plant proteins. We use corn leaves provided by one
of the researchers.
Kirsten

From: kmahovl@uoft02.utoledo.edu (Kirsten Mahovlich)


For an introductory electrophoresis lab, I have used a cryptomonad as a
source of proteins. The cryptomonad does not have a cell wall, so a
pellet of cells can be extracted with acetone, which removes the
non-polar pigments and lipids and precipitates the proteins. The
proteins are then solubilized with SDS buffer. In addition, the
cryptomonad possesses a pigmented phycobiliprotein. When run on a PAGE
gel with prestained markers, the phycobiliprotein can be seen, and the
molecular weight calculated, without staining.

Marvin Fawley
Department of Botany
North Dakota State University
fawley@plains.nodak.edu


George,
The biggest problem in dealing with plant cells (vs. animal) is
the cell wall. There are enzymes available which will digest the cell
wall and leave you with a protoplast to work with. I have not worked with
plant tissue culture for a few years, but the last time this was done we
bought enzymes from Japan (which is a lengthy process) and Sigma
chemicals. What you need are cellulase and pectinase to do a good job of
removing the cell wall. A good reference book is Bhojwani & Razdan Plant
Tissue Culture: Theory & Practice; Elsevier, 1989. If you need more
information let me know and i will try and help.

Good luck,
Mike Weber
Dept. of Biology
Carleton University
Ottawa, Ontario
Canada
mweber@ccs.carleton.ca

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