vacuole prep

Hermann Hofte hofte at VERSAILLES.INRA.FR
Tue Mar 17 05:37:18 EST 1992

This is a vacuole prep protocol that worked very well in our hands both
for tobacco and Arabidopsis protoplasts (see note added).
Vacuole prep on tobacco leaf protoplasts.

(Procedure adapted from Marie-Theres Hauser).

Prepare protoplasts. Make sure your protoplasts are completely digested 
(rounded shape) 

Make up 4% ficoll solution: mix 1 vol lysis medium with 1.5 vol vacuole 
buffer (3 ml/ gradient)
Add BSA (0.5% final) to lysis medium, prewarm at 42 C. 

2-5.106 pps in  glass tube, spin 5min, 500rpm.

Remove most of the SN, resuspend pps in remaining liquid.

Add swiftly 5ml prewarmed(420C) lysis buffer, pipet the solution 2-3 x up 
and down (wide bore pipet), transfer to small glass tube.

Check under microsope, vacuoles should have come out, chloroplasts 
should be spread all over the place. If lysis not complete, try repeating the 
up and down in pipet thing. 

layer 3 ml 4% ficoll solution on lysis buffer and 1 ml of vacuole buffer.
(4% ficoll solution made with 1 vol lysis buffer (no BSA) and 1.5 vol 
vacuole buffer)
20 min 3 K swing out (RC-3 or HB4), vacuoles should concentrate at the 
interphase between the 4 % ficoll and the vacuole buffer. Collect vacuoles 
with a pasteur pipet.

Lysis buffer: 	0.2 M Mannit				
 			10 % ficoll (400)				
			20 mM EDTA							
			2 mM DTT					
			10 mM HEPES				
			pH 8.0 NaOH.
			Add 0.5 % BSA before use, prewarm at 42 C.

Vacuole buffer:	0.6 M betain				
			10 mM HEPES pH 7.5 NaOH					
			protease inhibitor cocktail 
			(0.5mM PMSF, 0.1 microg/ml leupeptin, 10 -7 M 	
			1mM EDTA					
			150 ug/ml BSA

1. For Arabidopsis: make up 4 % ficoll solution with 1 vol lysis buffer and  
	 1.5 vol vacuole buffer with 0.5 M mannitol instead of 0.6 M betain.
2. Depending on the physiology of the plant the vacuoles can be more or 
less dense. Therefore you will often find vacuoles also at the interphase 
between the 10 % and the 4 % layer.               

good luck, Herman Hofte.

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