ICC/IF image of ab2900 stained human HeLa (Human epithelial cell line from cervix adenocarcinoma) cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab2900, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor^® 594 WGA was used to label plasma membranes (red).

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: EEA1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: NIH3T3 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab2900 observed at 162 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab2900 was shown to recognize EEA1 in wild-type HAP1 cells as signal was lost at the expected MW in EEA1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and EEA1 knockout samples were subjected to SDS-PAGE. Ab2900 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1 μg/ml 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-EEA1 antibody - Early Endosome Marker (ab2900) at 1 µg/ml

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate
Lane 4 : HeLa whole cell lysate
Lane 5 : HEK-293 whole cell lysate
Lane 6 : NIH 3T3 whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG H&L (HRP) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 160 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?
Additional bands at: 100 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 30 seconds

This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin (Lanes 1-3) or 3% Milk (Lanes 4-6) before being incubated with ab2900 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

Abcam recommends using milk as the blocking agent. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.

All lanes : Anti-EEA1 antibody - Early Endosome Marker (ab2900) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) nuclear lysate
Lane 2 : HeLa whole cell lysate
Lane 3 : A431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 4 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 5 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Alexa Fluor anti rabbit at 1/50000 dilution

Performed under reducing conditions.

Predicted band size: 160 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?
Additional bands at: 100 kDa, 41 kDa, 50 kDa. We are unsure as to the identity of these extra bands.



Fluorescence detection of secondary antibody.

Confocal microscopy of fixed primary cultures of rat hippocampal neurons (embryonic day 18) showing immunocytochemical labelling of rabbit polyclonal to EEA1 (ab2900, 1/200; Alexa Fluor 488 1/200; green) and monoclonal mouse anti-β.

ab2900 Rabbit polyclonal to EEA1 (1/500) was used in fixed HEK cells that had been transfected with Rab5-GFP. Fluorescence microscopy of fixed Rab5-transfected HEK cells showing (left to right) immunocytochemical labelling rabbit polyclonal to EEA1 ab2900 (1/500; Alexa Fluor^® 568; red), and Rab5-GFP (green) with their overlay (far right). Scale bar = 5µm. Showing little co-localisation as expected.

ab2900 staining mouse L-cells by ICC/IF.  Cells were formaldehyde fixed, permeabilized in Triton X-100 and incubated with ab2900 diluted 1/1000 for 1 hour at 37°C.  A Cy2® conjugated goat anti-rabbit antibody was used as the secondary.

See Abreview

All lanes : Anti-EEA1 antibody - Early Endosome Marker (ab2900) at 1/500 dilution

Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : HEK293 Whole Cell lysate with Human EEA1 peptide (ab14946) at 1 µg

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 160 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?



Lane 1 - 2 : EEA1 antibody - Early Endosome Marker (ab2900) at 1/500 dilution, HEK293 Whole Cell lysate at 20 ug Lane 1 : as above Lane 2 : EEA1 peptide (ab14946) at 1 ug Secondary Goat polyclonal to Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution Performed under reducing conditions. Predicted band size : 160kD Observed band size : 180kD (why is the actual band size different from the predicted?)

All lanes : Anti-EEA1 antibody - Early Endosome Marker (ab2900) at 1 µg/ml

Lane 1 : Xenopus laevis lysate
Lane 2 : Mouse 3T3 cell lysate
Lane 3 : Mouse brain cell lysate
Lane 4 : Mouse liver cell lysate
Lane 5 : Rat brain cell lysate
Lane 6 : Rat liver cell lysate
Lane 7 : Dog lysate
Lane 8 : CHO cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 160 kDa
Observed band size: 100,180 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

The Western blot shows that ab14944 reacts strongly with mouse 3T3 and CHO cell lysates. Weak cross-reactivity is seen with Xenopus, mouse brain, mouse liver, rat brain and dog lysates. The antibody does not appear to cross-react with rat liver lysate.