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MOLECULAR BIOLOGY: WORKING WITH DNA

PROTEIN EXPRESSION: LABELING

In Vivo Labeling of Plant Proteins and Immunoprecipitation

In Vivo Labeling of Plant Proteins and Immunoprecipitation
 
Overview
This protocol describes four suggestions on ways to metabolically label plant tissue followed by instructions on how to prepare the samples for immunoprecipitation.
 
Procedure
Various Methods for Radiolabeling Proteins with [35S] (see Hint #3)

A. Method #1

1. Paint [35S] Solution #1 on the underside of the leaf and incubate for 1 to 4 hr (see Hint #4).

B. Method #2

1. Paint [35S] Solution #2 on the underside of the leaf and incubate for approximately 4 hr.

C. Method #3

1. Cut a leaf with a sharp razor blade and add [35S]-TRANS Label (or use [35S]-Methionine) directly to the cut.

2. Incubate the leaves in the radioactive solution for approximately 1 hr (see Hint #5)

D. Method #4 Metabolic Labeling of Whole Plants

1. Put seedlings that have just germinated in small puddles of [35S] Sulfate Solution.

2. Incubate for 2, 3 or 4 days.

E. Homogenization and Immunoprecipitation

1. Freeze the tissue in Liquid Nitrogen.

2. Using a homogenizer with a glass pestle, add plant material to approximately 2 ml of ice-cold Homogenization Buffer.

3. Homogenize the tissue at 4°C.

4. Add 2 ml of Phenol and shake well (CAUTION! See Hint #1).

5. Centrifuge in a table-top centrifuge or equivalent at 2,000 X g for 5 min to separate the aqueous and organic phases.

6. Save the Phenol phase.

7. Add 5 volumes of 0.1 M Ammonium Acetate.

8. Precipitate overnight at -20°C.

9. Centrifuge the solution at 2,000 X g for 5 min to collect the protein pellet.

10. Decant the supernatant and add an equal volume of 80% Acetone to the protein pellet.

11. Mix the solution well.

12. Centrifuge at 2,000 X g for 5 min to collect the protein pellet. Discard the supernatant.

13. Allow the pellet to dry.

14. Resuspend the pellet in a solution of 1% (w/v) SDS, cap the tube loosely, and place the solution in a boiling water bath for 1 min, just long enough to finish dissolving the protein pellet.

15. Add 7 volumes of Tris/Triton Solution and mix well.

16. Centrifuge the solution briefly to pellet any insoluble material.

17. Save the radiolabeled supernatant.

18. Divide the supernatant into two equal samples. Add the antibody of interest to one aliquot. Add a control antibody to the second aliquot (see Hint #6).

19. Incubate for 1 hr at 4°C on a rotating platform.

20. When the binding is complete, precipitate the antibodies with 0.1 volume of 10% fixed Staphylococcus A cells (see Hint #7).

21. Wash the precipitates with Washing Buffer three times.

22. Resuspend the precipitates in Sample Buffer and boil for 3 min to release the immunoprecipitated proteins.

23. Centrifuge for 1 min in a microcentrifuge.

24. Load the samples onto a protein gel (see Protocol ID#455).

Solutions
[35S]-TRANS Label
[35S]-TRANS Label   1.4 mCi/ml [35S]-TRANS label (ICN) (CAUTION! See Hint #1 and Hint #2)
Only needed if using Method #3
[35S]-Methionine Solution #2
[35S]-Methionine Solution #2
Sample Buffer   10% Glycerol
0.05% Bromophenol Blue
2% SDS
50 mM Tris
100 mM DTT
Washing Buffer   5 mM EDTA
150 mM NaCl
50 mM Tris, pH 7.5
Tris/Triton Solution   50mM Tris, pH 7.8
1% (v/v) Triton-X-100
1% (w/v) SDS
80% (v/v) Acetone
[35S]-Methionine Solution #2
[35S]-Methionine Solution #2
[35S]-Methionine Solution #2   1.5 mCi/ml [35S]-Methionine (CAUTION! See Hint #1 and Hint #2)
Only needed if using Method #2 for labeling
0.02% (v/v) Tween 20
[35S] Solution #1
[35S] Solution #1
[35S] Solution #1
[35S] Solution #1
[35S] Solution #1   0.05% (v/v) Triton X-100
Only needed if using Method #1 for labeling
1.5 mCi/ml [35S] TRANS label (ICN) (CAUTION! See Hint #1 and Hint #2)
0.1 M Ammonium Acetate   Prepare in Methanol
Homogenization Buffer   0.1 M KCl
50 mM EDTA
*Add just before use
2 mM Phenylmethylsulfonyl Fluoride (PMSF)* (CAUTION! See Hint #1)
0.7 M Sucrose
0.5 M Tris, pH 9.4
2% (v/v) 2-Mercaptoethanol*
[35S] Sulfate Solution
[35S] Sulfate Solution
[35S] Sulfate Solution   Only needed if using Method #4
[35S] Sulfate (CAUTION! See Hint #1 and Hint #2)
[35S]-TRANS Label
 
BioReagents and Chemicals
Potassium Chloride
Sodium Chloride
Ammonium Acetate
[35S]-Methionine
DTT
Nitrogen, Liquid
Bromophenol Blue
SDS
Antibody
Methanol
Triton X-100
EDTA
Sucrose
[35S]-Sulfate
Glycerol
[35S]-Trans label
Fixed Staphylococcus A cells
Tween 20
Phenol
PMSF
2-Mercaptoethanol
Acetone
Tris
 
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. Trans label is a mixture of about 70% Methionine and about 15% Cysteine.

3. There are a number of different methods for radiolabeling leaf proteins (these are listed as Sections A through D).

4. Paint on the underside because of the presence of more stomata.

5. This method was found to work better than painting label on leaves, getting approximately 400,000 cpm total from one leaf.

6. The binding conditions will vary greatly from protein to protein. Determine the right binding conditions for your own application empirically. Make sure you have determined the immunoprecipitation dose response curve.

7. The contributor of this protocol suggests PANSORBIN cells from Calbiochem.