All lanes : Anti-Cyclin E2 antibody [EP454Y] (ab40890) at 1/10000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : CCNE2 knockout HAP1 whole cell lysate
Lane 3 : MCF7 whole cell lysate
Lane 4 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 50 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab40890 observed at 45 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab40890 was shown to react with CCNE2 in HAP1 wild-type cells in Western blot. Loss of signal was observed when CCNE2 knockout sample was used. HAP1 wild-type and CCNE2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween^®) before incubation with ab40890 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 10000 dilution and a 1 in 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^® 800CW) preabsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue sections labeling Cyclin E2 with purified ab40890 at 1/100 dilution (1.41 µg/ml). Heat mediated antigen retrieval was performed Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Cyclin E2 with purified ab40890 at 1:50 dilution (2.8 µg/ml). Cells were fixed in 100% Methanol and permeabilized with None. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Anti-Cyclin E2 antibody [EP454Y] (ab40890) at 1/1000 dilution (Purified) + Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 50 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?

ab40890 (purified) at 1/20 dilution (1 µg) immunoprecipitating Cyclin E2 in Jurkat whole cell lysate.
Lane 1 (input): Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10 µg
Lane 2 (+): ab40890 & Jurkat whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab40890 in Jurkat whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

ab40890 (unpurified) at 1/250 staining human breast carcinoma by immunohistochemistry, paraffin-embedded tissue.

Flow cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Cyclin E2 (right) with purified ab40890 at a 1/20 dilution. Cells were fixed with 90% ethanol and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution.

Left panel - Rabbit monoclonal IgG (ab172730).