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

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : CCNE1 (Cyclin E1) knockout HAP1 whole cell lysate

Lysates/proteins at 40 µg per lane.

Predicted band size: 47 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab33911 observed at 47 kDa. Red - 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 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.

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

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. 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.

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 (ab172730) instead of ab33911 in HeLa whole cell lysate.
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

Lane 1 : Anti-Cyclin E1 antibody [EP435E] (ab33911) at 1/1000 dilution (Purified)
Lanes 2-3 : Anti-Cyclin E1 antibody [EP435E] (ab33911) at 1/1000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : Human testis lysates
Lane 3 : Human placenta lysates

Lysates/proteins at 20 µg per lane.

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 why is the actual band size different from the predicted?

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

Blocking/Diluting buffer: 5% NFDM/TBST

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, 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).

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) (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.

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. 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.

See Abreview

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.

See Abreview