重组Anti-Cyclin E1抗体[EPR194] (ab133266)

Western blot: Anti-CCNE1 antibody [EPR194] (ab133266) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab133266 was shown to bind specifically to CCNE1. A band was observed at 45 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CCNE1 knockout cell line. To generate this image, wild-type and CCNE1 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Western blot: Anti-CCNE1 antibody [EPR194] (ab133266) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab133266 was shown to bind specifically to CCNE1. A band was observed at 47 kDa in wild-type MCF7 cell lysates with no signal observed at this size in CCNE1 knockout cell line ab286303 (knockout cell lysate AB300211). To generate this image, wild-type and CCNE1 knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1% Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Cyclin E1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: MCF7 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab133266 observed at 47 kDa. Red - loading control, ab9484, observed at 37 kDa.

Unpurified ab133266 was shown to recognize Cyclin E1 in wild-type cells as signal was lost at the expected MW in Cyclin E1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Cyclin E1 knockout samples were subjected to SDS-PAGE. Ab133266 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Cyclin E1 with Purified ab133266 at 1:1000 dilution (1.6μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 μg/ml). 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.

Intracellular Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling Cyclin E1 with purified ab133266 at 1/150 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Unpurified ab133266 staining Cyclin E1 in HeLa cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Samples were incubated with primary antibody (1/500 in PBS) for 1 hour at 22°C. ab150081, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody. Counterstained with DAPI.

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