Recombinant Anti-FAK antibody [EP695Y] (ab40794)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Rabbit (1:500) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Rabbit (1:500) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Publication protocol

Focal adhesions were isolated using an established protocol (21). In brief, cells were exposed to ice-cold 2.5 mM triethanolamine inducing a hypotonic shock for 3 min. By rigorous pipetting with cold water containing protease inhibitor tablets (complete mini EDTA-free, Roche (Basel, Switzerland)), the remaining cell bodies were removed by hydrodynamic force. For samples subjected to shear, the center of the coverslips was covered with Parafilm that had been circularly cut using a crafting punch device (The Punch Bunch, Temple, TX) and removed before shear application to ensure that all cells were subjected to a minimum shear of 600 dynes/cm2. FAs were collected with mRIPA buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1% Triton X-100, 1% Na-DOC, and 0.1% sodium dodecyl sulfate (SDS)) with 1 mM EGTA, 1 mM Na3VO4, 10 mM Na4P2O7, and 1 mM phenylmethanesulfonylfluoride for Western blots. Samples were run in 10% SDS-polyacrylamide gel electrophoresis (PAGE) gels at 150 V until proteins were separated and then transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA) to be run at 100 V for 1 h 15 min in the transfer apparatus (Bio-Rad). The membranes were washed in Buffer A (25 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween-20) 15% milk overnight at 4°C and then incubated for 2 h with the following antibodies: Vinculin (ab129002) at 1/10,000, focal adhesion kinase (FAK) (ab40794) at 1/500, Paxillin (ab32084) at 1/5000 (all from Abcam), GAPDH (MAB374) at 1/2500 (Millipore, Darmstadt, Germany), Fibronectin (R457) at 1/2000 (22), and Actin (JLA20) at 1/5000 (Millipore). After three 10-min washes with Buffer A, secondary goat antirabbit HRP (Bio-Rad) and anti-mouse HRP (Abcam) were used for incubation for 30 min. Immunoblots were visualized using ECL reagent (Thermo Fisher).

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Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-FAK antibody [EP695Y] (ab40794) below.

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