Anti-Fibronectin antibody (ab23750)

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

Tissue lysates of knockout and WT hearts were prepared via homogenization of atria or ventricles on ice in lysis buffer (150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10 mM Tris/HCl) with protease and phosphatase inhibitors (Roche-Applied Sciences; 100 mM NaF and 100 mM Na3VO4). Thirty micrograms of protein from lysates were resolved on a 10% SDS/PAGE gel when probing for Cx40 and Cx43 or a 4–20% gradient gel (Bio-Rad, Hercules, CA) when probing for N-cadherin, collagen I, and fibronectin, and then transferred to a nitrocellulose membrane using an iBlot Dry Blotting system (Invitrogen). Membranes were blocked in a 3% BSA/0.05% Tween-20 PBS solution (PBST) for 30 min at room temperature. Membranes were then probed using the following primary antibodies: rabbit anti-Cx43 (1:5000, Sigma, C 6219); goat anti-Cx40 (1:500, Santa Cruz Biotechnology, sc-20466); mouse anti-collagen 1 (1:500, Abcam, ab90395), mouse anti-fibronectin (1:500, Abcam, ab23750), mouse anti-N-cadherin (1:200, BD Signal Transduction, 610920), and mouse anti-GAPDH (Santa Cruz Biotechnology, sc-365062), diluted in blocking solution at 4°C overnight. Membranes were then washed three times for 5-min intervals with PBST and incubated with fluorescently tagged secondary anti-rabbit Alexa Fluor® 680 (1:5000, LI-COR Biosciences, ab175772) or anti-mouse IRdye 800 (1:5000, Rockland Immunochemicals, Inc., 610-132-003). Protein expression and densitometry analysis was determined using Odyssey Infrared Imaging System and software (LI-COR Biosciences). Samples were normalized to GAPDH loading controls; n=3 samples per group.

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Manufacturer protocol

Download the product protocol from Abcam for Anti-Fibronectin antibody (ab23750) below.

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