p95 NBS1 Antibody (monoclonal, 5D7A1)

• Catalog Number: orb865592
• Category: Antibodies
• Description: p95 NBS1 Antibody (monoclonal, 5D7A1)
• Species/Host: Mouse
• Clonality: Monoclonal
• Clone Number: 5D7A1
• Tested applications: FC, ICC, IF, IHC, WB
• Reactivity: Human, Mouse, Rat
• Isotype: Mouse IgG2a
• Immunogen: A synthetic peptide corresponding to a sequence at the C-terminus of human p95 NBS1 (714-745aa RKNTELEEWLRQEMEVQNQHAKEESLADDLFR), different from the related mouse sequence by three amino acids, and from the related rat sequence by five amino acids.
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 95 kDa
• UniProt ID: O60934
• Sensitivity: > 5000 cells
• 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: Apoptic death agonist antibody; Apoptotic death ag
• 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. Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Expiration Date: 12 months from date of receipt.

Western blot analysis of p95 NBS1 using anti-p95 NBS1 antibody (orb865592). 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 30 ug of sample under reducing conditions. Lane 1: human Jurkat whole cell lysates, Lane 2: human A549 whole cell lysates, Lane 3: rat brain tissue lysates, Lane 4: rat testis tissue lysates, Lane 5: mouse brain tissue 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-p95 NBS1 antigen affinity purified monoclonal antibody (Catalog # orb865592) 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 p95 NBS1 at approximately 95 kDa. The expected band size for p95 NBS1 is at 95 kDa.

IHC analysis of p95 NBS1 using anti-p95 NBS1 antibody (orb865592). p95 NBS1 was detected in a paraffin-embedded section of human testis cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml mouse anti-p95 NBS1 Antibody (orb865592) 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.

IF analysis of p95 NBS1 using anti-p95 NBS1 antibody (orb865592). p95 NBS1 was detected in an 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 5 μg/mL mouse anti-p95 NBS1 Antibody (orb865592) 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 A431 cells using anti-p95 NBS1 antibody (orb865592). Overlay histogram showing A431 cells stained with orb865592 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-p95 NBS1 Antibody (orb865592, 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 (Red line) was also used as a control.