CD59 Antibody(monoclonal, 3C10)

• Catalog Number: orb623829
• Category: Antibodies
• Description: CD59 Antibody(monoclonal, 3C10)
• Species/Host: Mouse
• Clonality: Monoclonal
• Clone Number: 3C10
• Tested applications: FC, ICC, IF, IHC, WB
• Reactivity: Human
• Isotype: Mouse IgG2b
• Immunogen: E.coli-derived human CD59 recombinant protein (Position: L26-N102). Human CD59 shares 47.1% amino acid (aa) sequence identity with rat CD59.
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Dilution range: Western blot, 0.1-0.5μg/ml, Human Immunohistochemistry (Paraffin-embedded Section), 2-5μg/ml, Human Immunocytochemistry/Immunofluorescence, 2μg/ml, Human Flow Cytometry, 1-3μg/1x106, Human
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 19 kDa
• UniProt ID: P13987
• 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: CD59 glycoprotein: 1F5 antigen; 20 kDa homologous
• 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 500μg/ml.
• Expiration Date: 12 months from date of receipt.

IF analysis of CD59 using anti-CD59 antibody (orb623829). CD59 was detected in immunocytochemical section of A431 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (orb90553) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL mouse anti-CD59 Antibody (orb623829) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse 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.

IF analysis of CD59 using anti-CD59 antibody (orb623829). CD59 was detected in immunocytochemical section of HeLa cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (orb90553) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL mouse anti-CD59 Antibody (orb623829) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse 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 A549 cells using anti-CD59 antibody (orb623829). Overlay histogram showing A549 cells stained with orb623829 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-CD59 Antibody (orb623829, 1μg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (5-10μg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1μg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Western blot analysis of CD59 using anti-CD59 antibody (orb623829). 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: human HeLa whole cell lysates; Lane 2: human U-87MG whole cell lysates; Lane 3: human PC-3 whole cell 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 mouse anti-CD59 antigen affinity purified monoclonal antibody (Catalog # orb623829) 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 (Catalog # orb90502) with Tanon 5200 system. A specific band was detected for CD59 at approximately 19KD. The expected band size for CD59 is at 19KD.

IHC analysis of CD59 using anti-CD59 antibody (orb623829). CD59 was detected in paraffin-embedded section of human colorectal 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 2μg/ml mouse anti-CD59 Antibody (orb623829) 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) (Catalog # orb90443) with DAB as the chromogen.