FAK Autophosphorylation Assay

Description
This kinase assay is meant to determine whether an agonist or event can influence the autophosphorylation of FAK. The addition of 1 l of polyGT to the kinase reaction mix will determine the activity of the enzyme against a substrate.

Procedure
A. Harvest

1. Grow the cells to confluence in 100 mm dishes. Stimulate according to requirements of the agonist. The dose and time must be determined empirically.

2. Wash the monolayers with ice-cold Wash Buffer. Add 1 ml of ice-cold Lysis Buffer (1X) to each dish.

3. Scrape the cells from the dish and transfer the cells to a microcentrifuge tube on ice.

4. Rock gently at 4C for 30 min.

5. Centrifuge in a microcentrifuge at maximum speed for 5 min and transfer the cleared lysate to a new tube.

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

7. Supernatants may be aliquoted and stored at -20?C.

B. Immunoprecipitation

1. Use 300 g of protein (from Section A Step #6), and normalize the sample volumes to 1 ml.

2. Preclear the lysate by adding 20 l of prepared Protein A-Sepharose (see Protocol on Immunoprecipitation).

3. Rock at 4C for 20 min.

4. Centrifuge at 4C for 10 min to pellet the Sepharose and then transfer the supernatant to a new tube.

5. Add 1 g of each anti-FAK antibody to each tube and rock at 4C for 1 to 2 hr.

6. Add 30 l of Protein A-Sepharose and rock for an additional 30 min.

7. Centrifuge 10 min at 4C at 5,000 X g to pellet the Sepharose.

8. Discard the supernatant and wash the pellet twice with Lysis Buffer.

9. Repeat the wash once with 1X UKB.

10. Resuspend the pellet in 10 l of 1X UKB and keep on ice.

C. Kinase Reaction

1. Make a Master Mix of the following reagents:

9.5 l of 1X UKB

0.5 l of -[32P]-ATP

Add 10 l of this mix to the pellet (from Step #B10).

2. Incubate at 37C for 20 min.

3. Add an equal volume of 2X Laemmli Sample Buffer and heat for 3 min at 95?C.

4. Electrophorese the samples on a 8% SDS-Polyacrylamide Gel (see Protocol on SDS-PAGE).

5. Transfer the gel to Nitrocellulose (see Protocol on Western Blotting).

6. Keep the Nitrocellulose Blot of the protein for Section D. Dry the gel between cellophane sheets and expose to film (see Protocol on Autoradiography) for analysis.

D. Antibody Detection of FAK

1. Follow the standard Western Protocol (see Protein Detection by Western Protocol).

2. Combine the following reagents to detect the bound secondary antibody :

66 l of NBT Solution

33 l of BCIP Solution

in 10 ml of Alkaline Phosphatase Buffer

Mix well.

3. Pour the mix over the blot at room temperature and develop until the bands are suitably dark.

4. Stop the reaction by rinsing the blot in PBS containing 20 mM EDTA.

5. Dry the blot and expose to film .

6. Quantitate the bands (by Phosphoimage analysis of densitometry) and determine the phosphorylation ratio.

Recipes
10 mg/ml Leupeptin (CAUTION! see Hint #1)
Store at -20C

BCIP 0.25 g BCIP in 5 ml 100% DMF
Make just before use and keep on ice

10 mg/ml Aprotinin (CAUTION! see Hint #1)
Store at -20C

NBT Solution Make just before use and keep on ice
0.25 g of NBT in 5 ml of 70% DMF

100 mM PMSF (CAUTION! see Hint #1)
Store at -20C

70% (v/v) Dimethylformamide (DMF) (CAUTION! see Hint #1)

Lysis Stock Buffer (2X) Filter Sterilize
20 mM Tris, pH 7.4
Store at 4C
300 mM NaCl
20% (v/v) Glycerol
2 mM EDTA
2 mM EGTA

Alkaline Phosphatase Buffer Filter Sterilize
100 mM NaCl
pH to 9.5.
5 mM MgCl2
Store at 4C
100 mM Tris, pH 9.5

Wash Buffer in PBS
1 mM Sodium Orthovanadate (Na3VO4)

-[32P]-ATP 10 Ci/l of -[32P]-ATP (5000 Ci/mmol, CAUTION! see Hint #1)

Universal Kinase Buffer (UKB) (5X) 50 mM PIPES, pH 7.4
50 mM MnCl2

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

Lysis Buffer (1X) 1 mM Na3VO4
1% (v/v) NP-40
Make just before use.
0.1% (w/v) DOC
1 mM PMSF
50% (v/v) Lysis Stock Buffer
25 g/ml Aprotinin
0.1% (w/v) SDS
25 g/ml Leupeptin

Laemmli Sample Buffer (2X) 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

Supplies

Tips
1.CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
(责任编辑：泉水)

搜索

热门标签:

FAK Autophosphorylation Assay