All lanes : HRP Anti-Ezrin antibody [EP886Y] (ab198522) at 1/5000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : EZR (Ezrin) knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 69 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

ab198522 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. Ab198522 and ab184095 (Mouse monoclonal [mAbcam 9484] to GAPDH - Loading Control (Alexa Fluor^® 680) loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/1000 dilution respectively. The loading control was imaged using the Licor Odyssey CLx prior to blots being developed with ECL technique.

IHC image of Ezrin staining in a section of formalin-fixed paraffin-embedded normal human lung*, performed on a Leica BOND. 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 ab198522 at 1/100 dilution for 15 mins at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative 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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

HRP Anti-Ezrin antibody [EP886Y] (ab198522) at 1/5000 dilution + HeLa whole cell lysate (ab150035) at 10 µg

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 69 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?


Exposure time: 18 seconds

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab198522 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.