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Janet Braam braam at RICE.EDU
Thu Jul 2 17:18:15 EST 1992

Following is a summary of replies I received regarding in vivo labelling of 
plant proteins and immunoprecipitation.  (Sorry for the delay in posting!)
There are several methods of 
labelling the proteins, one protocol for cell lysis and protein recovery, 
and two suggestions for carrying out the immunoprecipitation.

Much THANKS! to all who replied.  

Labelling with 35S:

1.  paint the 35S on leaves in a solution of 0.05% Triton X-100, preferably on
the underside because of the presence of more stomata. (Chris Sommerville)

2.  paint with 35S methionine in a solution of 0.02% Tween20, allow it to be
taken up for 4 hours.  (Sarah Gilmour)

3.  Cut leaf with sharp razor blade and place in 35S met/cys ("EXPRE" from NEN
or TRANS-label from ICN) for one hour.  The label is diluted to 1.4mCi/mL. 
Found that this method works better than painting label on leaves getting 400,00
0dpm total from one leaf.  (Carrie Schneider)

4.  place detached leaves in eppendorf tubes with a short vacuum infiltration 
(Plant Physiol. 88:731.  note error in M&M, use 20ul, not 209ul of H20.  (Tim

5.  for labeling whole plants, use 35S sulfate instead of labelled amino acids.  
Put seedlings just geminated in small puddles of S35 sulfate for 2, 3 or 4 days.
(Russell Malmberg)

Homogenization and Immunoprecipitation:

1.  Homogenization and Immunoprecipitation.  Freeze tissue in liquid nitrogen.  
Homogenize in 2mL of (0.7M sucrose, 0.5MTris 9.4, 50mM EDTA, 0.1M KCl, 2%
mercaptoethanol, 2mM PMSF: from Hurkman and Tanaka, Plant Physiol. 81:802, 1986). 
Use a glass homogenizer.  Add 2mL phenol, shake.  Spin to separate and retain phenol 
phase.  Add 5 volumes of 0.1M NH4OAc in methanol.  Precipitate o/n in freezer.  
Spin out proteins.  Wash pellet with 80% acetone and dry.  Resuspend in 1% SDS
and boil for 1 min.  Add 7 volumes of (50mM Tris 7.8, 1% triton-X-100), spin out
insolubles.  Add antibody to this .  Precipitate the antibody with 1% crosslinked
stapholococcus A cells.  Wash the precipitate with (50mM Tris 7.5, 150mM NaCl, 5 mM
EDTA).  Resuspend in Laemmli buffer and load gel.  (Carrie Schneider).

2.  Immunoprecipitations. any standard protocol will get you
started, but I would urge you to optimize each reagent.
We found that we could greatly reduce the amounts of killed
Staph A cells, the amounts of rabbit-anti-mouse, and the
initial amounts of the specific monocloncal we were using.
When you think you have each of these reasonably optimized,
do a dose response of the amonut of protein in the supernatant
and the amount in the pellet as a function of increasing
specific antibody.  This will give you a dose of your antibody
that should work for routine assays.
We particularly found that bridge antibodies (such as the
rabbit anti-mouse) are particularly important to optimize, since
bunnies and other animals usually contain antibodies that
recognize some plant epitopes.


Plant extract

Hepes:  50 mM Hepes 

Hepes-BSA:  50 mM Hepes, 1% BSA

Washed pansorbin cells (Calbiochem):
     Remove 55 ul pansorbin cells.
     Spin down, resuspend in 55 ul Hepes-BSA
     Repeat 3 times.
     Let sit on ice at least 1 hour.

Rabbit anti-mouse: dilute 10x into Hepes-BSA (need 10 ul)

Monoclonal:  dilute 100x into Hepes-BSA  (need 14 ul)
             dilute 10x into Hepes-BSA (need 13 ul)
             straight, undiluted (need 13 ul)

1.  Gently mix the following, then incubate on ice for 60

                A    B    C    D    E    F    G    H 

100x dil        0    1    3   10    0    0    0    0
10x dil         0    0    0    0    3   10    0    0
straight        0    0    0    0    0    0    3   10
oat extract    25   25   25   25   25   25   25   25
Hepes-BSA      25   24   23   21   19   17   15   13

2.  Add 1 ul diluted rabbit anti-mouse to each tube.
     Gently mix, then incubate on ice for 1 hour.
3.  Add 5 ul Pansorbin to each tube.
     Gently mix, then incubate on ice for 1 hour.
4.  Washing (4 times):
     Spin down, resuspend in 100 ul Hepes-BSA.
     Spin down, resuspend in 100 ul Hepes-BSA.
     Spin down, resuspend in 100 ul Hepes-BSA.
     Spin down, resuspend in 100 ul Hepes-BSA.
5.  Enzyme assay each fraction.

(Russell Malmberg)

Janet Braam 
braam at bioc.rice.edu

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