RAC1 Polyclonal Antibody

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	-Rabbit (1:1000)

Protocol tips
-Rabbit (1:1000)

Publication protocol

Total cell lysates were prepared in RIPA buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml PMSF, 10 µg/ml Na3VO4, aprotenin, leupeptin, pepstatin; 1 ml/100 mm dish). Lysates were passed through a needle 18 times to shear the DNA and were spun at 15,000 rpm (21,700 g) for 20 min. Protein estimations were performed on the supernatant (MicroBCA protein assay kit, Pierce, Rockford, IL) with BSA as a standard. 20 µg of protein per sample was separated on a 12% SDS polyacrylamide gel and electro-blotted onto Imobilon-P membranes. The blots were probed with monoclonal or polyclonal anti-Cx26, -Cx32 and -Cx43 antibodies (Cell Signaling Technology, Danvers, MA), followed by a secondary antibody conjugated to horseradish peroxidase (HRP; Cell Signaling) and then ECL plus reagent (Amersham Biosciences) prior to exposure to X-ray film (Kodak). The blots were stripped (100 mM 2-mercaptoethanol, 2% w/v SDS, 62.4 mM Tris-HCl, pH 6.7) for 30 min at 50°C with mild agitation and re-probed with actin antibody (Sigma). Cell lysates from migrating cells were prepared 24 or 48 h after stamp-wounding confluent monolayers. Antibodies for N-cadherin (cat. no. 33-3900, Thermo-Fisher, Waltham, MA), p120 catenin (cat. no sc-23872, Santa Cruz Biotechnology, Santa Cruz, CA) and Rac1 (cat. no. PAI-091, Thermo-Fisher, Waltham, MA) were used according to manufacturer's instructions.

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Manufacturer protocol

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