Anti-CD146/MCAM Antibody (monoclonal, 4C12)

• Catalog Number: orb654279
• Category: Antibodies
• Description: Anti-CD146/MCAM Antibody (monoclonal, 4C12). Tested in Flow Cytometry, IHC, IHC-F, WB applications. This antibody reacts with Human.
• Clonality: Monoclonal
• Species/Host: Mouse
• Isotype: Mouse IgG2a
• Conjugation: Unconjugated
• Reactivity: Human
• Form/Appearance: Lyophilized
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Immunogen: E.coli-derived human CD146 recombinant protein (Position: H59-A401). Human CD146 shares 73% amino acid (aa) sequence identity with both mouse and rat CD146.
• UniProt ID: P43121
• MW: 120 kDa
• Tested applications: FC, IHC, IHC-Fr, WB
• Application notes: Western blot, 0.1-0.5μg/ml, Human Immunohistochemistry (Paraffin-embedded Section), 0.5-1μg/ml, Human Immunohistochemistry (Frozen Section), 0.5-1μg/ml, Human Flow Cytometry (Fixed), 1-3μg/1x106 cells, Human. Add 0.2ml of distilled water will yield a concentration of 500ug/ml
• Antibody Type: Primary Antibody
• Clone Number: 4C12
• Storage: Maintain refrigerated at 2-8°C for up to 2 weeks. For long term storage store at -20°C in small aliquots to prevent freeze-thaw cycles.
• Alternative names: A32 antigen antibody; CD 146 antibody; CD146 antib
• Note: For research use only

Flow Cytometry analysis of SiHa cells using anti-CD146/MCAM antibody. Overlay histogram showing SiHa cells (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with mouse anti-CD146/MCAM Antibody (1 µg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (5-10 µg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1 µg/1x10^6) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

IHC analysis of CD146/MCAM using anti-CD146/MCAM antibody. CD146/MCAM was detected in frozen section of human placenta tissue. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 µg/ml mouse anti-CD146/MCAM Antibody overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.

IHC analysis of CD146/MCAM using anti-CD146/MCAM antibody. CD146/MCAM was detected in paraffin-embedded section of human mammary cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 µg/ml mouse anti-CD146/MCAM Antibody overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.

IHC analysis of CD146/MCAM using anti-CD146/MCAM antibody. CD146/MCAM was detected in paraffin-embedded section of human placenta tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 µg/ml mouse anti-CD146/MCAM Antibody overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.

IHC analysis of CD146/MCAM using anti-CD146/MCAM antibody. CD146/MCAM was detected in paraffin-embedded section of human rectal cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 µg/ml mouse anti-CD146/MCAM Antibody overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.

Western blot analysis of CD146/MCAM using anti-CD146/MCAM antibody. Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50 ug of sample under reducing conditions. Lane 1: human A375 whole cell lysates; Lane 2: human Hela whole cell lysates. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-CD146/MCAM antigen affinity purified monoclonal antibody at 0.5 µg/mL overnight at 4°C, then washed with TBS-0.1% Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for CD146/MCAM at approximately 120 KD. The expected band size for CD146/MCAM is at 72 KD.