IHC image of Ezrin staining in a section of formalin-fixed paraffin-embedded normal human lung performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab4069, 0.025ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

IHC image of Ezrin staining in mouse lung formalin fixed paraffin embedded tissue section. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab4069, 1µg/ml overnight at +4°C. An HRP-conjugated secondary (Ab97040, 1/1000 dilution) was used for 1hr at room temperature. The section was counterstained with haematoxylin and mounted with DPX.

IHC image of ab4069 staining in 10% formaldehyde fixed frozen tissue section of human placenta.

Non-specific protein-protein interactions were blocked using TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 3% (w/v) BSA for 1 hour at room temperature. The section was then incubated with ab4069 (1μg/ml dilution) in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA overnight at +4°C. The section was then incubated with ab150119 (Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 647)) and DAPI for 1 hour at room temperature.

The DAPI only control (no antibody) inset shows no autofluorescence, demonstrating that any Alexa Fluor^® 647 signal is derived directly from bound ab4069. 

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antibody concentrations and incubation times.

All lanes : Anti-Ezrin antibody [3C12] (ab4069) at 1 µg/ml

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : EZR (Ezrin) knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : MEF whole cell lysate
Lane 5 : NIH3T3 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 69 kDa

Lanes 1 - 5: Merged signal (red and green). Green - ab4069 observed at 81 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab4069 was shown to specifically react with Ezrin in wild-type HAP1 cells as signal was lost in EZR (Ezrin) knockout cells. Wild-type and EZR (Ezrin) knockout samples were subjected to SDS-PAGE. Ab4069 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Anti-Ezrin antibody [3C12] (ab4069) at 1/500 dilution + Mouse bEnd.3 whole cell lysate at 20 µg

Secondary
HRP-conjugated goat anti-mouse IgG polyclonal at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 69 kDa
Observed band size: 81 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

See Abreview

Overlay histogram showing SH-SY5Y cells stained with ab4069 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab4069, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.