P-Cadherin antibody

Antigen: P-Cadherin

Clonality: Monoclonal (99-2#82)

Host: Mouse

Reactivity: Human, Rat (Rattus)

Application: Western Blotting (WB), Immunoprecipitation (IP)

Catalog no.: ABIN131667

Additional Information

Immunogen: This antibody was purified from hybridoma (clone 99-2#82) supernatant using protein-A Sepharose. This hybridoma was established by fusion of mouse myeloma cell P3/NS1/1-Ag4-1 with Balb/c mouse splenocyte immunized with the fusion protein between full-length P-cadherin and maltose binding protein.

Isotype: IgG1  (Matching secondary antibodies)

Clone: 99-2#82

Description: Cadherins are a family of transmembrane glycoproteins involved in Ca 2+ -dependent cell-cell adhesion. Cadherins contribute to the growth and development of cells via the maintenance of tissue integrity and tissue architecture. They are expressed in a tissue specific manner and are required for assembly of cells into solid tissue. Individual cadherin molecules cooperate to form a linear cell adhesion zipper. In adhesion junctions, cadherins bind to ß- and .-catenins which in turn bind to a-catenin, an actin binding protein. In addition to roles in differentiation and tissue morphogenesis, cadherins also appear to play a significant role in modulating tumor invasion and metastasis. Placental cadherin (P-cadherin) is an 829 amino acid polypeptide localized in placenta while E-Cadherin and N-Cadherin are found in epithelial and neural tissues, respectively. P-cadherin is selectively expressed in basal and parabasal layers of stratified epithelia. It may be involved in the final stages of tumor progression in epidermal carcinoma. Changes in P-cadherin expression have been implicated in breast and melanocytic tumors.

Specificity: This antibody reacts with human and rat P-cadherin.

Application Details

Application Notes: SDS-PAGE & Western Blotting 1) Wash the cells 3 times with PBS and suspend with 10 volumes of cold Lysis buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol) containing appropriate protease inhibitors. Incubate it at 4 o C with rotating for 30 minutes, then sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 o C and transfer the supernatant to another tube. Measure the protein concentration of the supernatant and add the Lysis buffer to make an 8 mg/mL solution. 3) Mix the sample with equal volume of Laemmli’s sample buffer. 4) Boil the samples for 2 minutes and centrifuge. Load 10 µL of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 for 1 hour in a semi-dry transfer system. (Transfer Buffer - 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for specific transfer procedure. 6) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4°C. 7) Incubate the membrane with the anti-P-cadherin monoclonal antibody (5 µg/mL) diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 8) Wash the membrane with PBS (5 minutes x 6 times). 9) Incubate the membrane with the 1:10000 POD-conjugated anti-mouse IgG (MBL, code no. 330) diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 10) Wash the membrane with PBS (5 minutes x 6 times). 11) Wipe excess buffer from the membrane, then incubate it with appropriate chemiluminescence reagents for 1 minute. Remove extra reagent from the membrane by dabbing with a paper towel, and seal it in plastic wrap. 12) Expose to X-ray film in a dark room for 5 minutes. Develop the film as usual. The conditions for exposure and development may vary. Positive controls for Western blotting A431 031031-1 Western blotting: 5 µg/mL Immunoprecipitation: not tested.

Concentration: 1 mg/mL

Buffer: 100 µg IgG in 100 µL volume of PBS containing 50% glycerol, pH 7.2. Contains no preservatives.

Restrictions: For Research Use only

Publications

Publications: Wahl: "Generation of monoclonal antibodies specific for desmoglein family members." in: Hybridoma and hybridomics, Vol. 21, Issue 1, pp. 37-44, 2002 (PubMed).