WB; IF, Frozen; Immunohistochemistry: Antibody detects sparse Cxn45-IR in caudal artery and heart tissue (see Rummery, NM et al 2002 for more staining specifics). Antibody was used at 1:100 to 1:250, but Biosensis recommends optimal dilutions/concentrations should be determined by the end user. In the original work the specificity of the antibody was shown 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.
Western Blot: Antibody is not recommended for western blotting by Biosensis, however, it does react in westerns with Cxn 45 specific material. The authors report that the antibody develops numerous bands in westerns blots, only some of which are removed upon peptide treatment (see Rummery, NM et al 2002). The Cx45/354 antibody revealed the presence of a specific 45-kDa band in all tissues tested, although it was very weak in the arteries. A higher molecular weight band, which was blocked by peptide, was also seen in the brain (see Rummery, NM et al 2002, online Figure VIIB, -/+ peptide).
免疫原
A synthetic peptide (QAYSHQN NPHGPRE) as part of human Connexin-45 protein (aa: 354-367) conjugated to diphtheria toxoid has been used as the immunogen.