Recombinant 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, 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 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 ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

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 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.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Cyclin B1 with purified ab32053 at 1:400 dilution (1 µg/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.

Blocking and diluting buffer: 5% NFDM/TBST

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 (ab172730) instead of ab32053 in Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate

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

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. 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.

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

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.

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) (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.