BubR1/BUB1B Antibody (monoclonal, 5I7)

• Catalog Number: orb654276
• Category: Antibodies
• Description: BubR1/BUB1B Antibody (monoclonal, 5I7)
• Species/Host: Mouse
• Clonality: Monoclonal
• Clone Number: 5I7
• Tested applications: FC, ICC, IF, IHC, WB
• Reactivity: Human
• Isotype: Mouse IgG1
• Immunogen: E.coli-derived human BubR1/BUB1B recombinant protein (Position: K26-E448).
• 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), 0.5-1μg/ml, Human Immunocytochemistry/Immunofluorescence, 2μg/ml, Human Flow Cytometry, 1-3μg/1x106 cells, Human
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 130 kDa
• UniProt ID: O60566
• 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: Mitotic checkpoint serine/threonine-protein kinase
• 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 BubR1/BUB1B using anti-BubR1/BUB1B antibody (orb654276). 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 HEK293 whole cell lysates; Lane 3: human K562 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-BubR1/BUB1B antigen affinity purified monoclonal antibody (Catalog # orb654276) 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 BubR1/BUB1B at approximately 130KD. The expected band size for BubR1/BUB1B is at 120KD.

IHC analysis of BubR1/BUB1B using anti-BubR1/BUB1B antibody (orb654276). BubR1/BUB1B was detected in paraffin-embedded section of human ovarian 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 1μg/ml mouse anti-BubR1/BUB1B Antibody (orb654276) 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.

IHC analysis of BubR1/BUB1B using anti-BubR1/BUB1B antibody (orb654276). BubR1/BUB1B 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 1μg/ml mouse anti-BubR1/BUB1B Antibody (orb654276) 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.

IHC analysis of BubR1/BUB1B using anti-BubR1/BUB1B antibody (orb654276). BubR1/BUB1B 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 1μg/ml mouse anti-BubR1/BUB1B Antibody (orb654276) 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.

IHC analysis of BubR1/BUB1B using anti-BubR1/BUB1B antibody (orb654276). BubR1/BUB1B 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 1μg/ml mouse anti-BubR1/BUB1B Antibody (orb654276) 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.

Flow Cytometry analysis of HeLa cells using anti-BubR1/BUB1B antibody (orb654276). Overlay histogram showing HeLa cells stained with orb654276 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-BubR1/BUB1B Antibody (orb654276, 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.

IF analysis of BubR1/BUB1B using anti-BubR1/BUB1B antibody (orb654276). BubR1/BUB1B 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-BubR1/BUB1B Antibody (orb654276) 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.