Anti-EEA1 antibody [EPR4245] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-EEA1 antibody [EPR4245] - BSA and Azide free (AB239942)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109110).

• WB

Lab

Western blot - Anti-EEA1 antibody [EPR4245] - BSA and Azide free (AB239942)

This data was developed using the same antibody clone in a different buffer formulation (ab109110).

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 editedcell 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:

Western blot - Anti-EEA1 antibody [EPR4245] - Early Endosome Marker (<a href='/en-us/products/primary-antibodies/eea1-antibody-epr4245-early-endosome-marker-ab109110'>ab109110</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

EEA1 CRISPR/Cas9 edited HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human EEA1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-eea1-knockout-hela-cell-line-ab261822'>ab261822</a>)

Lane 3:

Daudi cell lysate at 20 µg

Lane 4:

SH-SY5Y cell lysate at 20 µg

Predicted band size: 162 kDa

Observed band size: 175 kDa

false