Anti-EEA1 antibody - Early Endosome Marker (ab2900)

ab2900 staining EEA1 in HepG2 cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab2900 at 5µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

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.

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.

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 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.

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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.