Connexin-40 (Cx40) Antibody

A synthetic peptide consisting of amino acids 254 to 270 of the C-terminus of rat Cx40 (Cx40/254) conjugated to diphtheria toxoid has been used as the immunogen.

Immunohistochemistry: Antibody detects Cxn 40 in rat tissues and arterial endothelial cells. The authors report that the density of Cx40 plaques was significantly greater in the caudal artery (CA) than in the thoracic aorta (ThA), whereas no such difference was seen for Cx37 and Cx43. Expression of Cx40 was absent from the media of both thoracic and caudal artery tissues (see Rummery, NM et al 2002 for more staining specifics) . Published Method: Unfixed 10 μm thick sections cryosections or lightly fixed (2% paraformaldehyde in 0.1 mol/L sodium phosphate buffer) whole mount sections have been tested, see Rummery, NM et al 2002) . Pretreatments include pre-incubation for 30 minutes in a blocking solution of 2% bovine serum albumin (BSA), 0.2% Triton-X in PBS, followed by primary antibody incubation. Original detection was via Cy3- conjugated anti-goat immunoglobulins (Jackson Immunoresearch Laboratories Inc, PA, 1:100) in 0.01% Triton-X in PBS, but other secondary conditions should work as well once optimized. In the original work the specificity of each antibody was tested by incubation either without primary antibody or with primary antibody that had previously been pre-incubated for 1 hour at room temperature with 10-fold excess by weight of the peptide against which the antibody was raised. (Adapted from Rummery, NM et al 2002) . Western Blot: Antibody is not recommended for Western blots, however, it does react in Westerns with Cxn 40 specific material. The authors report that the antibody develops numerous bands in Western blots, only some of which are removed upon peptide treatment (see Rummery, NM et al 2002) . The Cx40/254 antibody specifically recognized a band of 40 kDa from lung, caudal artery (CA), and thoracic aorta (ThA) but not liver (online Figure VIIA, +/- peptide) . In the lung, however, a band at 45 kDa also appeared to be reduced with Cxn 40 peptide addition. Published Method: Brain, heart, liver, lung, thoracic aorta and caudal arteries were removed from 5-6 week old Wistar rats and snap frozen in liquid nitrogen Tissues were ground under liquid nitrogen in a mortar and pestle and resuspended in 1mL of lysis buffer (1 mM NaHCO3 pH 7.05, 10 mM EDTA, 10 mM Iodoacetamide, 10 mM tetra-sodium pyrophosphate, 1 mM PMSF and 1 μg/mL each of antipain, aprotinin, pepstatin-A, chymostatin and leupeptin) . Tissues were further disrupted by grinding in a polytron blender. Unbroken cells and large debris were removed by centrifugation at 1000 g for 5 minutes at 4oC, the supernatant was then removed and centrifuged at 3000 g for 5 minutes. The pellet was discarded, and the supernatant centrifuged at 20000 g for 15 minutes at 4°C. The supernatant was discarded, and the membrane-enriched pellet was resuspended in lysis buffer. Protein concentration was measured using the Bio-Rad protein assay kit. Membrane-bound connexins were subsequently solubilized by incubation in 2x SDS sample buffer (5% SDS, 125 mM Tris-Cl (pH 6.8), 20% glycerol, 2 mM β- mercaptoethanol, 0.1% (w/v) bromophenol blue) for 60 minutes at 37°C. Aliquots containing 5 μg of protein were separated by SDS-PAGE on 12% polyacrylamide gels and blotted onto PVDF membranes. Blots were probed with sheep antibodies against Cx40 (1:1000, Cx40/254) and detected via ECL using anti-Goat poly HRP secondary antibodies 1:4000, 1 hr. (adapted from Rummery, NM et al 2002) .