Anti-Cyclin E1 antibody [EPR194]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Cyclin E1 antibody [EPR194] (AB133266)

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).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cyclin E1 antibody [EPR194] (AB133266)

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.

• WB

Lab

Western blot - Anti-Cyclin E1 antibody [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.

All lanes:

Western blot - Anti-Cyclin E1 antibody [EPR194] (ab133266) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

CCNE1 knockout HCT 116 cell lysate at 20 µg

Lane 3:

Wild-type MCF7 ab288560 cell lysate at 20 µg

Lane 4:

CCNE1 knockout MCF7 <a href='/en-us/products/cell-lines/human-ccne1-knockout-mcf7-cell-line-ab286303'>ab286303</a> cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 47 kDa

Observed band size: 45 kDa

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• WB

Lab

Western blot - Anti-Cyclin E1 antibody [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 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.

All lanes:

Western blot - Anti-Cyclin E1 antibody [EPR194] (ab133266) at 1/1000 dilution

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lane 2:

CCNE1 knockout MCF7 cell lysate at 20 µg

Lane 3:

HT-29 cell lysate at 20 µg

Lane 4:

PC-3 cell lysate at 20 µg

Observed band size: 47 kDa

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• WB

Lab

Western blot - Anti-Cyclin E1 antibody [EPR194] (AB133266)

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.

Lane 1:

Western blot - Anti-Cyclin E1 antibody [EPR194] (ab133266) at 1/1000 dilution

Lanes 1 - 4:

Western blot - Anti-Cyclin E1 antibody [EPR194] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/cyclin-e1-antibody-epr194-bsa-and-azide-free-ab208695'>ab208695</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Cyclin E1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

MCF7 whole cell lysate at 20 µg

Observed band size: 47 kDa

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• WB

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Western blot - Anti-Cyclin E1 antibody [EPR194] (AB133266)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Cyclin E1 antibody [EPR194] (ab133266) at 1/10000 dilution

Lane 1:

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

Lane 2:

JAR (Human placenta choriocarcinoma epithelial cell) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 47 kDa

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• ICC/IF

AbReview45361****

Immunocytochemistry/ Immunofluorescence - Anti-Cyclin E1 antibody [EPR194] (AB133266)

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.

This image is courtesy of an Abreview submitted by Kirk McManus

• WB

Unknown

Western blot - Anti-Cyclin E1 antibody [EPR194] (AB133266)

All lanes:

Western blot - Anti-Cyclin E1 antibody [EPR194] (ab133266) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

MCF7 cell lysate at 10 µg

Lane 3:

JAR cell lysate at 10 µg

Lane 4:

K562 cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 47 kDa

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