Hi Tamsin,

Further to my e-mail of Friday last, I've done some more digging for
more information on protein extraction. The new morsels of
information are pasted into the relevant sections of my previous
message.

> I just read your enquiry about plant protein extraction.
>
> As you can see, even being bed ridden doesn't prevent me from
> chipping in my tuppence worth.
>
> My work with plant proteins goes back to the days of PhD, too,
> working with Mimosa pudica.
>
> Here's a short procedure, unearthed from the deepest dungeons
> of my memory (assuming that you are interested in whole protein
> without the fats and not being fuzzy).
>
> (1) Freeze your chopped up plant material in liquid nitrogen.
> (2) Grind the frozen material using pestle and mortar (add liquid
> N2 if required).

(2a) Grind the the frozen material using a grinding buffer; something
like ice-cold TRIS-HCl (0.2 M, pH 8.5; 0.25 M sucrose; 1.0 mM
EDTA; 0.25 mM phenylmethylsulfonyl fluoride; 0.1 mM MgCl2).
Rule of thumb: for 500 mg of plant material use 2 x 0.75 mL of
grinding buffer (i.e. in two steps). By way of explanation, the EDTA
and PMSF are used to switch off protease activity.

(2b) Filter the slurry through nylon (a piece of tights for instance, or
a cheesecloth) and centrifuge at 4,000 g for 20 min, then re-
centrifuge at 50,000 g fro 45 min.

(3) Add MeOH / H2O / glacial HOAc (or trichloroacetic acid 1%) to
the supernatant to precipitate proteins. (Go to (4))

> (3) Homogenise the ground material in methanol : water : glacial
> acetic acid (80:15:5, v/v/v).
> (4) Centrifuge for at least 10 min at 20,000 to 30,000 g (30 min
> can't hurt).

(4a) You can check if all protein has been precipitated by heating
the supernatant to 100 C for 15 min. This should precipitate
whatever managed to stay in solution.

> (5) Wash the protein pellet with chloroform : methanol : water
> (60:25:15 v/v/v) for defatting / delipidating.

(5a) Alternatively, wash (extract) the precipitate with n-heptane) or
n-hexane) to remove fatty components.

> (6) Centrifuge some more at 25,000 g.
>
> The beauty of this method is that it can be used for your
> purposes without modification. (A) It does not introduce any
> reactive nitrogen, and (B) all the solvents are volatile and can be
> removed under vacuum.

Should I find more, I'll let you know.


Best wishes,

                         Wolfram



Dr. W. Meier-Augenstein, CChem MRSC
Senior Research Fellow

University of Dundee, Dept. of Anatomy & Physiology,
OMS, Small's Wynd, DUNDEE  DD1 4HN, United Kingdom

Tel.: +44-(0)1382-34/5124, /4574, /4968
Fax:  +44-(0)1382-34/5514

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URL: http://www.dundee.ac.uk/anatphys/wma/meieraug.htm


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