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Intact chloroplasts from Arabidopsis

daemon at net.bio.net daemon at net.bio.net
Mon May 18 11:21:39 EST 1998


Dear Aranetters,

Some time ago I asked for a more effective method to isolate intact
chloroplast from Arabidopsis. Here are the answers.  The best way surely is
via preparing protoplasts first - which was too time-consuming for my
studies, so I never tried this. Thanks again.

Henning Pluecken

Institut fuer Entwicklungs- und
Molekularbiologie der Pflanzen

Heinrich-Heine-Universitaet

D-40225, Duesseldorf, Germany

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When I was making chloroplast many years ago I found that the most
useful breaking medium contained 10 mM EDTA.  Once the tissue is
broken the plastids should be pelleted at low speed (300 g) and then
resuspended in standard bufffers containing magnesium.  I found that
when protoplasts were ruptured in this medium I got >95% intactness
whereas when divalent ions were present I got essentially zero
intactness.  I think the basic method was described in Kunst, L., J.
Browse, C. R. Somerville.  Altered regulation of lipid biosynthesis
in a mutant of Arabidopsis deficient in chloroplast glycerol
phosphate acyltranferase activity. Proc. Natl. Acad. Sci. USA
85,4143-4147 (1988). Good luck,

Chris Somerville
260 Panama Street
Stanford CA 94305

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I have found that different chloroplasts require a different
concentration of mannitol/sorbitol to remain intact.  Unfortunately,
I do not know the optimum concentration necessary for
Arabidopsis chloroplasts, but if you have been isolating them
using spinach methodology, it may be that the breakage
problem is due to osmotic shock.

Good luck in figuring this out!
Margaret Hollingsworth
(SUNY at Buffalo)

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Protokoll:
-	Cut leaves from young plants (grown at short days 8 to 12h light)
-	Store the leaves at 4=83C in the dark for one night
-	Grind tissue 2-3 sec in 1-5 vol grinding buffer in a Waring blender
	(do the whole isolation at 4=83C)
-	Filter homogenate through one layer of Miracloth
-	Centrifuge filtrate for 10 min at 1200xg at 2=83C, use slow breaking
-	Resuspend pellet in washing buffer
-	Layer suspension on a Percoll step gradient:
	- 5 ml 80% Percoll, 10 ml 40% percoll in washing buffer, for spinach
	- 5 ml 55% Percoll, 10 ml 35% percoll in washing buffer, for
arabidopsis
	(solution from ~50 g of leaves per gradient, up to 100g for spinach)
-	Centrifuge for 20 min at 3000xg in a swinging bucket rotor at 2=83C,
stop 	without breaking
-	Remove the intact chloroplast from the 80/40 (55/35) interphase
-	Dilute chloroplasts 6-7 times with washing buffer
-	Centrifuge for 5 min at 3000xg, use slow breaking
-	Repeat washing if necessary
-	Resuspend pellet in 5-10 =B5l washing buffer per gram tissue, examine by
	light microscopy
-	Freeze in 50 =B5l aliquots at -80=83C

Grinding buffer (pH 7.8):		Washing buffer (pH 7.8):

0.33 M Sucrose			0.33 M Sucrose
30 mM NaPPi			10 mM MOPS-KOH
0.1% BSA

5x washing buffer (pH 7.8, for gradient):

1.65 M Sucrose
50 mM MOPS-KOH

adapted from: Douce and Joyard, 1982

Christian Roth
IBPV
Batiment de Biologie
Universite de Lausanne
CH-1015 Lausanne
Christian.Roth at ibpv.unil.ch
Tel: +41 21 692 42 34
=46ax: +41 21 692 41 95
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