Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

Unpurified ab40839 showing positive staining in Prostatic carcinoma tissue.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

Unpurified ab40839 showing positive staining in Normal kidney tissue.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

Unpurified ab40839 showing positive staining in Hepatocellular carcinoma tissue.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Ezrin with Purified ab40839 at 1 : 500 dilution (1.6 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

Unpurified ab40839 showing positive staining in Breast carcinoma tissue.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

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.

• IP

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Immunoprecipitation - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

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.

All lanes:

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

Predicted band size: 69 kDa

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• WB

Supplier Data

Western blot - 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.

All lanes:

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

Predicted band size: 69 kDa

false

• WB

Lab

Western blot - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

Western blot : Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker ab40839 staining at 1/5000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 76 kDa in Wild-type A549 cell lysates with no signal observed at this size in EZR knockout A549 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

Lanes 1 - 3:

Western blot - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (ab40839) at 1/5000 dilution

Lanes 1 - 3:

Western blot - Anti-Ezrin antibody [EP886Y] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/ezrin-antibody-ep886y-bsa-and-azide-free-ab239832'>ab239832</a>) at 1/5000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

Western blot - Human EZR knockout A549 cell line (<a href='/en-us/products/cell-lines/human-ezr-knockout-a549-cell-line-ab300934'>ab300934</a>) at 20 µg

Lane 3:

HeLa at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 76 kDa

Observed band size: 76 kDa

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• WB

Lab

Western blot - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

All lanes:

Western blot - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (ab40839) at 1/20000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysates at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 69 kDa

Observed band size: 72 kDa

false

• WB

Unknown

Western blot - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (AB40839)

All lanes:

Western blot - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (ab40839) at 1/50000 dilution

All lanes:

Hela cell lysate at 10 µg

Predicted band size: 69 kDa

Observed band size: 72 kDa

false