Connexin 43 Antibody #3512

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Rabbit (1:1000)

Protocol tips
-Rabbit (1:1000)

Publication protocol

Western blots were performed on whole-cell extracts obtained by vortexing ESCs in cell extraction buffer (catalog number FNN0011; Life Technologies), followed by protein determination using the Thermo Scientific-Pierce BCA protein assay kit (catalog number PI-23227; Thermo-Fisher Scientific). Total protein (60 μg) was loaded onto NuPAGE Novex 4%–12% Bis-Tris protein gels and then transferred to polyvinyl difluoride membranes and blocked with 5% skim milk in PBS. Specific proteins were detected using polyclonal or monoclonal rabbit antibodies to the following human proteins: Cx43 (GJA1, 1:1000 dilution, catalog number 3512; Cell Signaling Technology); glycodelin-A (PAEP; 1:1000 dilution, catalog number ab103264; Abcam); CD10 (1:5000 dilution, catalog number ab79423; Abcam); fibronectin (1:1000 dilution, catalog number ab2413; Abcam). The following antibodies were supplied in the EMT antibody sampler kit: zonula occludens-1 (ZO1), β-catenin, N-cadherin, E-cadherin, vimentin (1:1000 dilution, catalog number 9782; Cell Signaling Technology); and discoidin domain receptor 2 (DDR2) (1:1000 dilution, catalog number 12133; Cell Signaling Technology). After overnight labeling with the primary antiserum, blots were then incubated with a secondary goat antirabbit or antimouse antibody (1:200000; catalog numbers 31460 and 31461; Pierce Biotechnology Inc) linked to horseradish peroxidase. The immunoreactive bands were visualized by the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech).
Blots were washed, reprobed with mouse monoclonal antihuman β-actin antibodies (1:1000 dilution, catalog number 31430; Sigma), and developed in an identical manner to ensure even loading. SeeBlue Plus2 prestained molecular weight standards (catalog number LC5925; Life Technologies), which are not detected by ECL, were used to calibrate the migration of proteins on the original gel and verify the identity of immunopositive bands. The size of each band is indicated in the figures. For quantification of proteins in the Western blots, the films were digitized on a flatbed scanner and Image J software (National Institutes of Health, Bethesda, Maryland) was used to integrate the density of each band. Data are presented as ratios of the MET or EMT markers normalized to β-actin band intensity.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Western blotting CX43 using Connexin 43 Antibody #3512 from Cell Signaling Technology.

Manufacturer protocol

Download the product protocol from Cell Signaling Technology for Connexin 43 Antibody #3512 below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Western blotting CX43 using Connexin 43 Antibody #3512 from Cell Signaling Technology. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.