Anti-Cyclin B1抗体[V152] (ab72)

ab72 staining Cyclin B1 in human tonsil tissue - formalin-fixed paraffin-embedded section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab72, 5 µg/ml, for 15 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Lanes:
Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: Cyclin B1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: HEK293 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab72 observed at 55 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab72 detected the expected band for Cyclin B1 in wild type HAP1 cells and the band was not seen in Cyclin B1 knockout HAP1 cells. Wild-type and Cyclin B1 knockout samples were subjected to SDS-PAGE. Ab72 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/10000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Flow cytometry analysis of HeLa cells stained with ab72 (green line). Cell harvesting/tissue preparation method - Trypsin/EDTA. Sample Buffer: Complete Media then PBS. Fixation: Formaldehyde. Permeabilization: 90% methanol. Dilution: 1/50. Incubation time: 1 hour at 23°C, diluent: PBS/10% goat serum. Secondary antibody: Goat anti-mouse FITC at 1/1000 dilution. 100000 cells were stained, non-specific IgG antibody - negative control (purple histogram)

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Lanes 1-9 shows the Cyclin B1 wave as HeLa cells enter and exit mitosis. HeLa cells were synchronised in the early S phase by double thymidine block then released to synchronously enter mitosis. Time points were taken at the points indicated.

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate loaded at 25 µg per lane. ab72 used at 1/500 in reducing conditions in conjunction with goat anti mouse (HRP) 1/20,000.

This image is courtesy of an Abreview submitted on 12 September 2005. We do not have any further information relating to this image.

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HeLa (Human epithelial cell line from cervix adenocarcinoma) cells stained for Cyclin B1 using ab72 at a dilution of 1/500 in ICC/IF. Secondary used is a Fluorescin-conjugated Goat Anti-Mouse IgG. Primary antibody incubated for 12 hours at +4°C in TBS. Blocked using 1% BSA for 10 minutes at 25°C. Cells were permeabilized with Tween-20.

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