RPA70 RPA1 Antibody (monoclonal, 11H4)

• Catalog Number: orb507562
• Category: Antibodies
• Description: Anti-RPA70 RPA1 Antibody (monoclonal, 11H4). Tested in Flow Cytometry, IF, IHC, WB applications. This antibody reacts with Human, Monkey, Mouse.
• Clonality: Monoclonal
• Species/Host: Mouse
• Isotype: Mouse IgG2b
• Conjugation: Unconjugated
• Reactivity: Human, Monkey, Mouse
• Form/Appearance: Lyophilized
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Purification: Immunogen affinity purified.
• Immunogen: A synthetic peptide corresponding to a sequence at the C-terminus of human RPA70, different from the related mouse sequence by three amino acids.
• UniProt ID: P27694
• MW: 12 kDa
• Tested applications: FC, IF, IHC, WB
• Application notes: Western blot, 0.1-0.5μg/ml Immunohistochemistry (Paraffin-embedded Section), 0.5-1μg/ml Immunofluorescence, 2μg/ml Flow Cytometry (Fixed), 1-3μg/1x106 cells. Add 0.2ml of distilled water will yield a concentration of 500ug/ml
• Cross Reactivity: No cross-reactivity with other proteins.
• Antibody Type: Primary Antibody
• Clone Number: 11H4
• 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: Replication protein A 70 kDa DNA-binding subunit;
• Note: For research use only

Flow Cytometry analysis of A431 cells using anti-RPA70 antibody. Overlay histogram showing A431 cells (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-RPA70 Antibody (1 µg/1x10^6 cells) for 30 min at 20°C. DyLight488 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 (Red line) was also used as a control.

IF analysis of RPA70 using anti-RPA70 antibody. RPA70 was detected in immunocytochemical section of A549 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2 µg/mL mouse anti-RPA70 Antibody overnight at 4°C. DyLight488 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.

IHC analysis of RPA70 using anti-RPA70 antibody. RPA70 was detected in paraffin-embedded section of human intestinal 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-RPA70 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 RPA70 using anti-RPA70 antibody. RPA70 was detected in paraffin-embedded section of human lung 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-RPA70 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 RPA70 using anti-RPA70 antibody. RPA70 was detected in paraffin-embedded section of human lung 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-RPA70 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 RPA70 using anti-RPA70 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: HELA whole cell lysates, Lane 2: MCF-7 whole cell lysates, Lane 3: K562 whole cell lysates, Lane 4: COS-7 whole cell lysates, Lane 5: Caco-2 whole cell lysates, Lane 6: A549 whole cell lysates, Lane 7: HEPG2 whole cell lysates, Lane 8: PC-3 whole cell lysates, Lane 9: HEPA1-6 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-RPA70 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 1:5000 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 RPA70 at approximately 12KD. The expected band size for RPA70 is at 12KD.