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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

ENZYMATIC ASSAY: OTHER

FAK AUTOPHOSPHORYLATION ASSAY

FAK Autophosphorylation Assay Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

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

Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

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 4°C 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 μg of protein (from Section A Step #6), and normalize the sample volumes to 1 ml. 2. Preclear the lysate by adding 20 μl of prepared Protein A-Sepharose (see Protocol on Immunoprecipitation). 3. Rock at 4°C for 20 min. 4. Centrifuge at 4°C for 10 min to pellet the Sepharose and then transfer the supernatant to a new tube. 5. Add 1 μg of each anti-FAK antibody to each tube and rock at 4°C for 1 to 2 hr. 6. Add 30 μl of Protein A-Sepharose and rock for an additional 30 min. 7. Centrifuge 10 min at 4°C 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 μl of 1X UKB and keep on ice. C. Kinase Reaction 1. Make a Master Mix of the following reagents: 9.5 μl of 1X UKB 0.5 μl of γ-[32P]-ATP Add 10 μl of this mix to the pellet (from Step #B10). 2. Incubate at 37°C 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 μl of NBT Solution 33 μl 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 (see Protocol on Autoradiography). 6. Quantitate the bands (by Phosphoimage analysis of densitometry) and determine the phosphorylation ratio.

Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

100 mM PMSF    (CAUTION! see Hint #1) Store at -20°C 70% (v/v) Dimethylformamide (DMF)    (CAUTION! see Hint #1) Lysis Stock Buffer (2X)    Filter Sterilize 20 mM Tris, pH 7.4 Store at 4°C 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 4°C 100 mM Tris, pH 9.5 Wash Buffer    in PBS 1 mM Sodium Orthovanadate (Na3VO4) γ-[32P]-ATP    10 μCi/μl of γ-[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 μg/ml Aprotinin 0.1% (w/v) SDS 25 μg/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 10 mg/ml Leupeptin    (CAUTION! see Hint #1) Store at -20°C 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 -20°C NBT Solution    Make just before use and keep on ice 0.25 g of NBT in 5 ml of 70% DMF

BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

Aprotinin Glycerol BCIP NBT 2-Mercaptoethanol Dimethylformamide (DMF) Manganese Chloride Deoxycholate DTT Orthovanadate Alkaline Phosphatase Conjugated Secondary Antibody Leupeptin PMSF Tris NP-40 Potassium Phosphate, Monobasic Sodium Phosphate, Dibasic EGTA PIPES Potssium Chloride Magnesium Chloride EDTA Protein A-Sepharose Primary Antibody gamma-[32P]-ATP Sodium Chloride Bromophenol Blue SDS

Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

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

   

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