Recombinant Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (ab40839)

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Ezrin knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: HCT116 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab40839 observed at 75 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab40839 was shown to specifically react with Ezrin in wild-type HAP1 cells as signal was lost in Ezrin knockout cells. Wild-type and Ezrin knockout samples were subjected to SDS-PAGE. Ab40839 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/5000 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast carcinoma tissue sections labeling Ezrin with Purified ab40839 at 1:250 dilution (3.28 µg/ml). Heat mediated antigen retrieval was performed Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human bladder carcinoma tissue sections labeling Ezrin with Purified ab40839 at 1:250 dilution (3.28 µg/ml). Heat mediated antigen retrieval was performed Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Ezrin with Purified ab40839 at 1/800 dilution (1 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

ab40839 (purified) at 1:40 dilution (2µg) immunoprecipitating Ezrin in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab40839 & HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab40839 in HeLa whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Overlay histogram showing SH-SY5Y cells stained with upurifiedab40839 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab40839, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in SH-SY5Y cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions. 

ICC/IF image of unpurified ab40839 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab40839 at 1/100 dilution overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Unpurified ab40839 at a 1:100 dilution staining Ezrin in human colon carcinoma tissue.

Fluorescent immunohistochemical analysis of paraffin-embedded human kidney carcinoma tissue using unpurified ab40839. Green-Ezrin red-PI

Unpurified ab40839 showing positive staining in Breast carcinoma tissue.

Unpurified ab40839 showing positive staining in Prostatic carcinoma tissue.

Unpurified ab40839 showing positive staining in Hepatocellular carcinoma tissue.

Unpurified ab40839 showing positive staining in Normal kidney tissue.