CD146/Mcam Antibody

• Catalog Number: orb654343
• Category: Antibodies
• Description: CD146/Mcam Antibody
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: ELISA, FC, IF, IHC, WB
• Reactivity: Mouse, Rat
• Isotype: Rabbit IgG
• Immunogen: E.coli-derived mouse CD146/Mcam recombinant protein (Position: E27-E621).
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Dilution range: Western blot, 0.1-0.25μg/ml, Mouse, Rat Immunohistochemistry (Paraffin-embedded Section), 0.5-1μg/ml, Mouse, Rat Immunofluorescence, 2μg/ml, Mouse Flow Cytometry, 1-3μg/1x106 cells, Mouse, Rat Direct ELISA, 0.1-0.5μg/ml, Mouse
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 120 kDa
• UniProt ID: Q8R2Y2
• Storage: Store at -20˚C for one year from date of receipt. After reconstitution, at 4˚C for one month. It can also be aliquotted and stored frozen at -20˚C for six months. Avoid repeated freeze-thaw cycles.
• Alternative names: Cell surface glycoprotein MUC18; Gicerin; Melanoma
• Note: For research use only
• Application notes: Tested Species: In-house tested species with positive results. Other applications have not been tested. Optimal dilutions should be determined by end users. Add 0.2ml of distilled water will yield a concentration of 500ug/ml.
• Expiration Date: 12 months from date of receipt.

Western blot analysis of MCAM using anti-MCAM antibody (orb654343). 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 50ug of sample under reducing conditions. Lane 1: rat lung tissue lysates, Lane 2: rat ovary tissue lysates, Lane 3: rat PC-12 whole cell lysates, Lane 4: mouse lung tissue lysates, Lane 5: mouse spleen tissue lysates, Lane 6: mouse thymus tissue lysates, Lane 7: mouse kidney tissue lysates, Lane 8: mouse ovary tissue lysates, After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-MCAM antigen affinity purified polyclonal antibody (Catalog # orb654343) at 0.25 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-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # orb90503) with Tanon 5200 system. A specific band was detected for MCAM at approximately 120KD. The expected band size for MCAM is at 120KD.

IHC analysis of MCAM using anti-MCAM antibody (orb654343). MCAM was detected in paraffin-embedded section of mouse intestine 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 1μg/ml rabbit anti-MCAM Antibody (orb654343) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90444) with DAB as the chromogen.

IHC analysis of MCAM using anti-MCAM antibody (orb654343). MCAM was detected in paraffin-embedded section of mouse intestine 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 1μg/ml rabbit anti-MCAM Antibody (orb654343) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90444) with DAB as the chromogen.

IHC analysis of MCAM using anti-MCAM antibody (orb654343). MCAM was detected in paraffin-embedded section of rat spleen 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 1μg/ml rabbit anti-MCAM Antibody (orb654343) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90444) with DAB as the chromogen.

IF analysis of MCAM using anti-MCAM antibody (orb654343). MCAM was detected in paraffin-embedded section of mouse intestine 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 2μg/mL rabbit anti-MCAM Antibody (orb654343) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of C6 cells using anti-MCAM antibody (orb654343). Overlay histogram showing C6 cells stained with orb654343 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-MCAM Antibody (orb654343, 1μg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (5-10μg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Flow Cytometry analysis of Neuro-2a cells using anti-MCAM antibody (orb654343). Overlay histogram showing Neuro-2a cells stained with orb654343 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-MCAM Antibody (orb654343, 1μg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (5-10μg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.