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.

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. 

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.

See Abreview

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.