Monday, October 27, 2003

Description
This protocol uses mitogen-activated protein/ERK kinase (MEK) to activate the extracellular-signal-regulated (Erk) mitogen-activated protein (MAP) kinases upon agonist binding to receptors.

Procedure
A. Harvest

1. Place confluent cells in Serum-Free Media 48 hrs before the start of the experiment.

2. Stimulate the cells (the amount of time is dependent on the agonist and must be determined empirically).

3. To harvest, wash the monolayers with 3 ml of ice-cold 0.15 M NaCl and scrape into 1 ml of ice-cold Lysis Buffer.

4. Transfer the lysate to a 1.5 ml microcentrifuge tube and incubate on ice for 30 min.

5. Centrifuge 5 min at maximum speed in a microcentrifuge.

6. Transfer the cleared lysate to a new tube.

7. Determine the protein concentration (see Protocol on Protein Quantitation)

8. Supernatants can be made into aliquots and stored at -20(C until needed.

B. Immunoprecipitation

1. For each kinase assay, start with 500 g total protein (may need more or less depending on the cell type). Bring the volume to 1 ml with Lysis Buffer.

2. To each tube add:

20 l of Protein-A Sepharose (see protocol on Immunoprecipitation)

10 l of anti-raf antibody

3. Incubate for 2 hr at 4(C on a rotating platform.

4. Centrifuge for 20 min at 10,000 rpm (setting 10 on a microcentrifuge) and wash pellet twice with 1 ml of Lysis Buffer.

5. Wash once with Kinase Buffer (without DTT)

C. Kinase Reaction

1. Resuspend the pellet in:

25 l of purified MEK

5 l of ATP Mix

2. Incubate for 30 min at room temperature.

3. Add 6 l of Laemmli Sample Buffer (4X) and heat for 3 min at 95(C.

4. Load 20 l on a 10% SDS-Polyacrylamide Gel (see Protocol on SDS-PAGE) for analysis.


Recipes
Laemmli Sample Buffer (1X) 2% (w/v) 2-Mercaptoethanol or 3.1% (w/v) DTT
125 mM Tris
0.01% (w/v) Bromophenol Blue
20% (v/v) Glycerol
4% (w/v) SDS


ATP Mix 1 mCi/ml -[32P]-ATP (5000 Ci/mmol)
in Kinase Buffer
0.6 mM ATP


Kinase Buffer (10X) *Make this solution with and without the DTT
Adjust pH to 7.3.
10 mM MgCl2
1 mM DTT*
150 mM NaCl
25 mM -Glycerophosphate
25 mM HEPES, pH 7.5


Lysis Buffer (CAUTION, see Hint #1) 1% (v/v) Triton X-100
5 g/ml Aprotinin
Add the last five ingredients just before use. Keep solution ice-cold.
1 mM Sodium Orthovanadate (Na3VO4)
20 mM Tris, pH 7.5
0.25 mg/ml Pefabloc
0.1% (v/v) 2-Mercaptoethanol
10 mM Benzimidine
1 mM PMSF
150 mM NaCl
2 mM EDTA
10% (v/v) Glycerol



Supplies


Tips
1.CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.