Anti-Cyclin E1 antibody [EP435E]

14 product images

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  Western blot - Anti-Cyclin E1 antibody [EP435E] (ab33911)

  False colour image of Western blot: Anti-Cyclin E1 antibody [EP435E] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab33911 was shown to bind specifically to Cyclin E1. A band was observed at 47 kDa in wild-type HAP1 cell lysates with no signal observed at this size in CCNE1 knockout cell line. To generate this image, wild-type and CCNE1 knockout HAP1 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 (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

  All lanes: Western blot - Anti-Cyclin E1 antibody [EP435E] (ab33911) at 1/1000 dilution

  Lane 1: Wild-type HAP1 cell lysate at 20 µg

  Lane 2: CCNE1 knockout HAP1 cell lysate at 20 µg

  Lane 3: Jurkat cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 47 kDa

  Observed band size: 47 kDa

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  Immunoprecipitation - Anti-Cyclin E1 antibody [EP435E] (ab33911)

  Purified ab33911 at 1/30 dilution (2ug) immunoprecipitating Cyclin E1 in HeLa whole cell lysate.
  Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate (10μg)
  Lane 2 (+): ab33911 + HeLa whole cell lysate.
  Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab33911 in HeLa whole cell lysate.
  VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) (1/1000) was used for Western blotting.
  Blocking Buffer and concentration: 5% NFDM/TBST.
  Diluting buffer and concentration: 5% NFDM/TBST.
  Observed band size: 50 kDa

  All lanes: Immunoprecipitation - Anti-Cyclin E1 antibody [EP435E] (ab33911)

  Predicted band size: 47 kDa

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  Western blot - Anti-Cyclin E1 antibody [EP435E] (ab33911)

  Lanes 1 - 2: Merged signal (red and green). Green - ab33911 observed at 47 kDa. Red - loading control, Anti-GAPDH antibody [mAbcam 9484] - Loading Control ab9484, observed at 37 kDa.
  

  ab33911 was shown to recognize CCNE1 in wild-type HAP1 cells as signal was lost at the expected MW in CCNE1 (Cyclin E1) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CCNE1 (Cyclin E1) knockout samples were subjected to SDS-PAGE. ab33911 and Anti-GAPDH antibody [mAbcam 9484] - Loading Control 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 Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  This image was generated using the unpurified format of the antibody.

  All lanes: Western blot - Anti-Cyclin E1 antibody [EP435E] (ab33911) at 1/1000 dilution

  Lane 1: Wild-type HAP1 whole cell lysate at 40 µg

  Lane 2: CCNE1 (Cyclin E1) knockout HAP1 whole cell lysate at 40 µg

  Predicted band size: 47 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-Cyclin E1 antibody [EP435E] (ab33911)

  Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labeling Cyclin E1 (green) with purified ab33911 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Nuclei were counterstained with DAPI (blue).
  

  Secondary Only Control: PBS was used instead of the primary antibody as the negative control.

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  Western blot - Anti-Cyclin E1 antibody [EP435E] (ab33911)

  Cyclin E1 is highly expressed in testis and placenta which is described in PMID: 9840943.
  

  Blocking/Diluting buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-Cyclin E1 antibody [EP435E] (ab33911) at 1/1000 dilution

  Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

  Lane 2: Human testis lysates at 20 µg

  Lane 3: Human placenta lysates at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

  Predicted band size: 47 kDa

  Observed band size: 50 kDa

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  This image is courtesy of a customer review submitted by Kirk McManus.

  Immunocytochemistry/ Immunofluorescence - Anti-Cyclin E1 antibody [EP435E] (ab33911)

  Immunocytochemistry/Immunofluorescence analysis of HeLa cells labeling Cyclin E1 with ab33911 at 1/500 dilution. Cells were fixed in paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Staining with ab33911 at 1/500 was carried out for 1 hour at 22°C in PBS buffer. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, a Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed secondary antibody, was used at 1/200 dilution. DAPI was used to counterstain.
  

  This image was generated using the unpurified format of the antibody.

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  This image is courtesy of an anonymous customer review.

  Immunocytochemistry/ Immunofluorescence - Anti-Cyclin E1 antibody [EP435E] (ab33911)

  ab33911 staining Cyclin E1 in HeLa cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 2% BSA for 45 minutes at room temperature. Samples were incubated with primary antibody (1/300 in PBS + 2% BSA) for 14 hours at 4°C. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.
  

  This image was generated using the unpurified format of the antibody.

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  Flow Cytometry (Intracellular) - Anti-Cyclin E1 antibody [EP435E] (ab33911)

  Overlay histogram showing MCF7 cells stained with ab33911 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab33911, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in MCF7 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
  

  This image was generated using the unpurified format of the antibody.

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  Western blot - Anti-Cyclin E1 antibody [EP435E] (ab33911)

  Western blot: Anti-CCNE1 antibody [EPR26497-64] (ab33911) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab33911 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 [EP435E] (ab33911) 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 Human wild-type MCF7 cell line ab288560 cell lysate at 20 µg

  Lane 4: CCNE1 knockout MCF7 Human CCNE1 knockout MCF7 cell line ab286303 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

  Performed under reducing conditions.

  Predicted band size: 47 kDa

  Observed band size: 45 kDa

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  Western blot - Anti-Cyclin E1 antibody [EP435E] (ab33911)

  Western blot: Anti-CCNE1 antibody [EP435E] (ab33911) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab33911 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 Human CCNE1 knockout MCF7 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 [EP435E] (ab33911) 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

  Developed using the ECL technique.

  Performed under reducing conditions.

  Observed band size: 47 kDa

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  Flow Cytometry (Intracellular) - Anti-Cyclin E1 antibody [EP435E] (ab33911)

  Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Cyclin E1 with Purified ab33911 at 1/30 dilution (10 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin E1 antibody [EP435E] (ab33911)

  Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling Cyclin E1 with ab33911 at 1/2000 (0.12 μg/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on the human placenta. The section was incubated with ab33911 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
  Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
  
  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin E1 antibody [EP435E] (ab33911)

  Immunohistochemical analysis of paraffin-embedded (A) Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) cell pellets (B)CCNE1 KO HAP1 cell pellets tissue labeling Cyclin E1 with ab33911 at 1/2000 (0.12 μg/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on (A) Wild-type HAP1 cell pellets, no staining on (B) CCNE1 KO HAP1 cell pellets.The section was incubated with ab33911 for 30 mins at room temperature.
  The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
  Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
  
  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin E1 antibody [EP435E] (ab33911)

  mmunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling Cyclin E1 with ab33911 at 1/2000 (0.12 μg/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on the human colon carcinoma. The section was incubated with ab33911 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
  Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
  
  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins