Anti-EEA1 抗体 [EPR4245] - Early Endosome Marker (ab109110)

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 CRISPR/Cas9 edited 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 CRISPR/Cas9 edited cell line ab261822 (CRISPR/Cas9 edited cell lysate ab256897) lane below 175kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and EEA1 CRISPR/Cas9 edited 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?