Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Early Endosome Marker 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 - ab109110 observed at 162 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab109110 was shown to recognize Early Endosome Marker in wild-type HAP1 cells as signal was lost at the expected MW in Early Endosome Marker knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Early Endosome Marker knockout samples were subjected to SDS-PAGE. Ab109110 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

Immunocytochemistry/Immunofluorescence analysis of JAR (human placenta choriocarcinoma epithelial) cells labelling EEA1 with ab109110 at a dilution of 1/250. Cells were fixed with 4% paraformaldehye and permeabilized with 0.1% TritonX-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG was used as the secondary antibody at a dilution of 1/1000. Counterstained with DAPI and ab195889, anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594), at a dilution of 1/200.

Image shows cytoplasmic staining in JAR cell line.

All lanes : Anti-EEA1 antibody [EPR4245] - Early Endosome Marker (ab109110) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : EEA1 knockout HeLa cell lysate
Lane 3 : Daudi cell lysate
Lane 4 : SH-SY5Y cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 162 kDa
Observed band size: 175 kDa why is the actual band size different from the predicted?

Lanes 1- 4: Merged signal (red and green). Green - ab109110 observed at 175 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109110 was shown to react with EEA1 in wild-type HeLa cells in western blot. The band observed in knockout cell line ab261822 (knockout cell lysate ab256897) lane below 175kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and EEA1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109110 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated 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) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-EEA1 antibody [EPR4245] - Early Endosome Marker (ab109110) at 1/10000 dilution

Lane 1 : COS-1 cell lysate
Lane 2 : NIH 3T3 cell lysate
Lane 3 : C6 cell lysate
Lane 4 : HeLa cell lysate
Lane 5 : Jurkat cell lysate
Lane 6 : JAR cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 162 kDa
Observed band size: 170 kDa why is the actual band size different from the predicted?