JNK/SAP Kinase Assay

Monday, October 27, 2003

Description
JNK/SAP Kinase Assay

Procedure
A. Stimulation and Harvesting of Cells

1. Grow cells to confluence in 100 mm culture dishes.

2. After cells reach confluence, grow in serum-free media for up to 48 hours.

3. Stimulate cells (see Hint #2).

4. Wash the cell monolayer with 3 ml of ice-cold 150 mM NaCl.

5. Add 1 ml of ice-cold Lysis Buffer.

6. Place cell culture dishes on a tabletop shaker at 4C for 30 min.

7. Transfer the total cell lysate from the cell culture dish to a microcentrifuge tube.

8. Centrifuge the cell lysate in a microcentrifuge at maximum speed for 5 min.

9. Transfer the supernatant to new microcentrifuge tube.

10. Determine the protein concentration (see protocol on Quantification of Protein; also see Hint #3)

B. Immunoprecipitation of JNK1 and JNK2

1. To a microcentrifuge tube add the following:

1.5 l of JNK1 Antibody

1.5 l of JNK2 Antibody

20 l of Protein-A Agarose Resin

80 l of PBS

Mix gently

2. Incubate with gently mixing at 4C for 2 hr.

3. Centrifuge in a microcentrifuge to pellet the JNK-Coupled-Protein-A Agarose Resin for 20 min at maximum speed and discard the supernatant.

4. To the Protein-A Agarose Resin add 1 ml of PBS and mix gently.

5. Pellet the Protein-A Agarose Resin as in Step #B3.

6. For each kinase assay, combine 20 l of JNK-Coupled-Protein-A Agarose Resin and approximately 500 g of total protein (prepared in Section A) and bring the final volume to approximately 1 ml with Lysis Buffer (see Hint #4).

7. Incubate at 4C for 20 min on a rotating mixing wheel.

8. Centrifuge in a microcentrifuge to pellet the JNK-Coupled-Protein-A Agarose Resin for 20 min at maximum speed and discard the supernatant.

9. To wash the JNK-Coupled-Protein-A Agarose Resin : add 1 ml of Lysis Buffer, mix gently and centrifuge as in Step #B8. Repeat wash one more time.

10. To wash the JNK-Coupled-Protein-A Agarose Resin by add 1 ml of 1X Kinase Buffer, mix gently and centrifuge as in Step #B8.

C. Kinase Reaction

1. Resuspend the JNK-Coupled-Protein-A Agarose Resin in the following:

25 l of GST-Reaction Buffer

5 l of ATP Mix

2. Incubate for 20 min at 30C.

3. Add 6 l of 4X Laemmli Buffer and incubate for 3 min at 95C.

4. Centrifuge in a microcentrifuge to pellet the JNK-Coupled-Protein-A Agarose Resin for 20 min at maximum speed and SAVE the supernatant.

5. Resolve 20 l of the supernatant on a 10% SDS-PAGE (see protocol on SDS-PAGE of Proteins).

6. Dry the gel between cellophane sheets with Annarap.

7. Expose for between 3 to 16 hours to a phosphoimaging plate (see Hint #5),

Recipes
Lysis Buffer 40 l of 0.5 M EDTA (final concentration 2 mM)
7.88 ml of ddH2O
375 l of 4 M NaCl (final concentration 150 mM)
100 l of 100 mM Sodium Orthovanadate (Na3VO4) (final concentration 1 mM)
100 l of PMSF (final concentration 1 mM)
Add the following just before use
50 l of Aprotinin (final concentration 5 g/ml)
200 l of 20 mM Tris, pH 7.5 (final concentration 20 mM)
Store at room temperature for no more than 1 week
Mix well and keep solution ice-cold until used
40 l of Leupeptin (final concentration 4 g/ml)
250 l of 1 M -Glycerophosphate (final concentration 25 mM)
100 l of Triton X-100 (final concentration 1% (v/v))
1 ml of Glycerol (final concentration 10%v/v))

PBS pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl

JNK2 Antibody Anti-JNK2 (FL) (Santa Cruz Biotech)

JNK1 Antibody Anti-JNK1 (C17) (Santa Cruz BioTech)

2 mM ATP, pH 7.0

10 mCi/ml -[32P]-ATP 10 mCi/ml -[32P]-ATP (5000 Ci/mmol; also CAUTION see Hint #1)

0.5 M DTT

1 M MgCl2

1 M HEPES, pH 7.4

100 mM PMSF 100 mM PMSF (CAUTION see Hint #1)
Aliquot in 105 l volume and store at 4C
in 100% Ethanol

Laemmli Sample Buffer (4X) 40% (v/v) Glycerol
0.02% (w/v) Bromophenol Blue
4% (w/v) 2-Mercaptoethanol or 3.1% (w/v) DTT
8% (w/v) SDS
250 mM Tris

150 mM NaCl

GST-Reaction Buffer Prepare just before use
0.8 g/ l GST-Jun (Amino Acids 1 through 165)
in 1X of Reaction Buffer

ATP Mix For ten reactions:
32.5 l of ddH2O
5 l of 10 mCi/ml -[32P]-ATP
7.5 l of 2 mM ATP, pH 7.0
5 l of 10X Reaction Buffer

Kinase Buffer (10X) 250 l of 1 M MgCl2 (final concentration 250 mM)
10 l of 100 mM Sodium Orthovanadate (Na3VO4) (final concentration 1 mM)
250 l of 1 M HEPES, pH 7.4 (final concentration 250 mM)
250 l of 1 M -Glycerophosphate (final concentration 250 mM)
220 l of ddH2O

100 mM Sodium Orthovanadate Aliquot in 105 l volume and store at -20C
100 mM Sodium Orthovanadate (Na3VO4)

Leupeptin Aliquot in 45 l volume and store at -20C
1 mg/ml Leupeptin

Reaction Buffer (10X) 250 l of 1 M MgCl2 (final concentration 250 mM)
10 l of 100 mM Sodium Orthovanadate (Na3VO4) (final concentration 1 mM)
250 l of 1 M -Glycerophosphate (final concentration 250 mM)
250 l of 1 M HEPES, pH 7.4 (final concentration 250 mM)
*Add just before use
20 l of 0.5 M DTT (final concentration 10 mM)*
220 l of ddH2O

Aprotinin Aliquot in 55 l volume and store at -20C
1 mg/ml Aprotinin

1 M -Glycerophosphate 1 M -Glycerophosphate
Store at -20C

4 M NaCl

0.5 M EDTA

1 M Tris, pH 7.5

Supplies

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

2. The dose and amount of time for stimulation of cells is dependent on the agonist of choice and therefore must be determined empirically.

3. If the protein supernatant is not going to be used immediately, the supernatant can be aliquoted (sample will be kinased using approximately 500 g total protein per sample) and stored at -20C.

4. The amount of total protein added to each reaction may need to be more or less depending on the cell type.

5. The time of exposure to a phosphoimaging plate id dependent on the amount of radiolabel incorporated.

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JNK/SAP Kinase Assay