Anti-Cyclin B1 antibody [Y106]

10 product images

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  Immunoprecipitation - Anti-Cyclin B1 antibody [Y106] (ab32053)

  ab32053 (purified) at 1:20 dilution (2μg) immunoprecipitating Cyclin B1 in Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate.
  
  Lane 1 (input): Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10μg
  Lane 2 (+): ab32053 & Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate
  Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab32053 in Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate
  

  For western blotting, VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used for detection at 1:1000 dilution.
  Blocking and diluting buffer: 5% NFDM/TBST.

  All lanes: Immunoprecipitation - Anti-Cyclin B1 antibody [Y106] (ab32053)

  Predicted band size: 48 kDa

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  Western blot - Anti-Cyclin B1 antibody [Y106] (ab32053)

  Lane 1: Wild-type HAP1 whole cell lysate (20 μg)
  Lane 2: CCNB1 knockout HAP1 whole cell lysate (20 μg)
  Lane 3: HeLa whole cell lysate (20 μg)
  Lanes 1 - 3: Merged signal (red and green). Green - ab32053 observed at 55 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
  

  Unpurified ab32053 was shown to specifically react with CCNB1 in wild-type HAP1 cells. No band was observed when CCNB1 knockout samples were examined. Wild-type and CCNB1 knockout samples were subjected to SDS-PAGE. ab32053 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/3000 dilution and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-Cyclin B1 antibody [Y106] (ab32053)

  Predicted band size: 48 kDa

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  Flow Cytometry (Intracellular) - Anti-Cyclin B1 antibody [Y106] (ab32053)

  Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Cyclin B1 with purified ab32053 at 1/400 dilution (1μg/ml) (Right). Cells were fixed with 80% Methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Left). Cells were pre-treated with 20μg/ml RNaseA for 30min to minimize the binding between PI and RNA.Then intracellular stained with ab32053 and PI.
  

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  Immunocytochemistry/ Immunofluorescence - Anti-Cyclin B1 antibody [Y106] (ab32053)

  Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Cyclin B1 with Purified ab32053 at 1:100 dilution. Cells were fixed in 100% Methanol. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

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  Western blot - Anti-Cyclin B1 antibody [Y106] (ab32053)

  Blocking and diluting buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-Cyclin B1 antibody [Y106] (ab32053) at 1/50000 dilution

  Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

  Lane 2: HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates at 15 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 48 kDa

  Observed band size: 58 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin B1 antibody [Y106] (ab32053)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human cervical carcinoma tissue sections labeling Cyclin B1 with Purified ab32053 at 1:250 dilution (1.47 μg/ml). Heat mediated antigen retrieval was performed using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

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  This image is courtesy of a customer review submitted by Stephanie Hilss.

  Immunocytochemistry/ Immunofluorescence - Anti-Cyclin B1 antibody [Y106] (ab32053)

  Unpurified ab32053 staining Cyclin B1 in the U2OS cell line from human by ICC/IF (Immunocytochemistry/immunofluorescence).Cells were fixed with formaldehyde,permeabilized with 1% Triton X-100 in PBS and blocked with 1% BSA for 1 hour at 37°C.Alexa Fluor® 594-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin B1 antibody [Y106] (ab32053)

  Unpurified ab32053 at a 1:100 dilution staining Human cyclin B1 in human skin carcinoma, using Immunohistochemistry, Paraffin Embedded Tissue.

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  Flow Cytometry (Intracellular) - Anti-Cyclin B1 antibody [Y106] (ab32053)

  Overlay histogram showing Jurkat cells stained with unpurified ab32053 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32053, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.
  

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  Western blot - Anti-Cyclin B1 antibody [Y106] (ab32053)

  All lanes: Western blot - Anti-Cyclin B1 antibody [Y106] (ab32053) at 1/20000 dilution

  Lane 1: Hela cell lysate.

  Lane 2: Jurkat cell lysate.

  Predicted band size: 48 kDa