MA5-13862

antibody from Invitrogen Antibodies

Targeting: EZR VIL2

Provider product page for MA5-13862

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [77]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [5]
Immunohistochemistry [3]
Other assay [10]

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Product number: MA5-13862 - Provider product page
Provider: Invitrogen Antibodies
Product name: Ezrin Monoclonal Antibody (3C12)
Antibody type: Monoclonal
Antigen: Recombinant protein fragment
Description: MA5-13862 targets Ezrin in IHC (P), ICC, IP, IF, and WB applications and shows reactivity with Bovine, Hamster, Human, Marsupial, mouse, Non-human primate, and Rat samples. The MA5-13862 immunogen is a recombinant fragment of human ezrin corresponding to aa 362-585.
Reactivity: Human, Mouse, Rat, Bovine, Hamster
Host: Mouse
Isotype: IgG
Antibody clone number: 3C12
Vial size: 500 µL
Concentration: 0.2 mg/mL
Storage: 4° C

EZR protein structure - MA5-13862 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of VIL2 was performed with 10 µg of HeLa cells transfected with Transfection Reagent alone (Lane 1), 100nM Non-Targeting control siRNA (Lane 2), or 100nM siRNA against VIL2 (Lane 3). Proteins were resolved using a NuPAGE® Novex 4-12% Bis-Tris Gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), and a protein size ladder. Proteins were wet transferred to a Pierce Nitrocellulose Membrane (Product # 88025) OR Pierce PVDF Membrane (Product # 88518) and blocked with Pierce Starting Block T20 (PBS) Blocking Buffer (Product # 37539) for 1 hour at room temperature. VIL2 was detected at ~ 81 kDa using VIL2 Mouse monoclonal antibody (Product # MA5-13862) diluted in Pierce Starting Block T20 (PBS) Blocking Buffer 4°C overnight on a rocking platform. Pierce Goat Anti-Mouse (Product # 31437) HRP-Conjugated Antibodies at a 1:2500 dilution were used and chemiluminescent detection was performed using Pierce Supersignal West Dura Maximum Sensitivity Substrate (Product # 37071). Relative density of the bands normalized to GAPDH (36 kDa). VIL2 Antibody (Product # MA5-13862) confirms silencing of VIL2 expression.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: CRISPR-Cas9 mediated genome editing ofEzrin (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR864181_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of Ezrin was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) and HeLa Cas9 cells transduced with Ezrin Lentiviral sgRNA (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-Ezrin Monoclonal Antibody (3C12) (Product # MA5-13862) using 0.5 µg/mL dilution and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177 1:5,000 dilution).Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific toEzrin (Fig (b)).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry analysis of Ezrin in U2OS cells. Cells fixed with 4% paraformaldehyde were permeabilized with 0.1% Triton X-100 (Product # 28314) in PBS for 15 minutes at room temperature and blocked with 2% BSA in PBST (Product # 37525) for 30 minutes at room temperature. Cells were treated with Peroxidase Suppressor (Product # 35000), probed with an Ezrin monoclonal antibody (Product # MA5-13862) at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBST, and incubated with an HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Product # 31431) at a dilution of 1:1000 for 30 minutes at room temperature. Chromogenic detection was performed using Metal Enhanced DAB Substrate Kit (Product # 34065). Images were taken on a Zeiss Axio Observer microscope at 20X magnification (x1.6 Optovar ~ 32X).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry analysis of Ezrin in U2OS cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 (Product # 28314) in TBST (Product # 28376) for 30 minutes at room temperature and blocked with Universal Blocker (from Product # 03-6000) for 30 minutes at room temperature. Cells were probed with an Ezrin monoclonal antibody (Product # MA5-13862) at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBST, and incubated with an alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) secondary antibody (Product # 31320) at a dilution of 1:250 for 30 minutes at room temperature. Chromogenic detection was performed using 1-Step NBT/BCIP (Product # 34042). Images were taken on a Zeiss Axio Observer microscope at 20X magnification (x1.6 Optovar ~ 32X).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Phalloidin (orange) and Ezrin (green) in A431 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA (Product # 37525) in PBS + 0.1% Triton X-100 for 30 minutes at room temperature. Cells were probed with an Ezrin monoclonal antibody (Product # MA5-13862) at a dilution of 1:75 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:250 for 30 minutes at room temperature. Actin was stained with DyLight 550 Phalloidin (Product # 21835) at a dilution of 1:120 (2.5 units/mL final concentration) and nuclei (blue) were stained with Hoechst (Product # 62249) at a concentration of 1 µg/mL for 30 minutes. Images were taken on a Zeiss Axio Observer Z1 microscope at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Phalloidin (magenta) and Ezrin (green) in A431 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA (Product # 37525) in PBS + 0.1% Triton X-100 for 30 minutes at room temperature. Cells were probed with an Ezrin monoclonal antibody (Product # MA5-13862) at a dilution of 1:75 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:250 for 30 minutes at room temperature. Actin was stained with DyLight 633 Phalloidin (Product # 21840) at a dilution of 1:120 (2.5units/mL final concentration) and nuclei (blue) were stained with Hoechst (Product # 62249) at a concentration of 1 µg/mL for 30 minutes. Images were taken on a Zeiss Axio Observer Z1 microscope at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Phalloidin (dark red) and Ezrin (green) in A431 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA (Product # 37525) in PBS + 0.1% Triton X-100 for 30 minutes at room temperature. Cells were probed with an Ezrin monoclonal antibody (Product # MA5-13862) at a dilution of 1:75 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:250 for 30 minutes at room temperature. Actin was stained with DyLight 680 Phalloidin (Product # 21839) at a dilution of 1:120 (2.5 units/mL final concentration) and nuclei (blue) were stained with Hoechst (Product # 62249) at a concentration of 1 µg/mL for 30 minutes. Images were taken on a Zeiss Axio Observer Z1 microscope at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on paraffin-embedded human breast cancer tissue sections. To expose target proteins, heat-induced epitope retrieval was performed using 10mM sodium citrate (pH 6.0) buffer for 20 minutes at 95ºC. Following antigen retrieval, tissues were blocked in 3% BSA (Product # 37525) in PBST for 30 minutes at room temperature and then probed with an Ezrin monoclonal antibody (Product # MA5-13862) at a dilution of 1:100 for 1 hour in a humidified chamber. Tissues were washed extensively with PBS/0.025% Tween-20 (Product # 28314) and endogenous peroxidase activity quenched with Peroxidase Suppressor (Product # 35000) for 30 minutes at room temperature. Detection was performed using an HRP-conjugated goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:500 followed by colorimetric detection using Metal Enhanced DAB Substrate Kit (Product # 34065). Images were taken on a Zeiss Axiovision microscope at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Formalin-fixed, paraffin-embedded human lung stained with Ezrin antibody using peroxidase-conjugate and DAB chromogen. Note cell membrane staining of bronchio-epithelial cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on paraffin-embedded human colon cancer tissue sections. To expose target proteins, heat-induced epitope retrieval was performed using 10mM sodium citrate (pH 6.0) buffer for 20 minutes at 95ºC. Following antigen retrieval, tissues were blocked in 3% BSA (Product # 37525) in PBST for 30 minutes at room temperature and then probed with an Ezrin monoclonal antibody (Product # MA5-13862) at a dilution of 1:100 for 1 hour in a humidified chamber. Tissues were washed extensively with PBS/0.025% Tween-20 (Product # 28314) and endogenous peroxidase activity quenched with Peroxidase Suppressor (Product # 35000) for 30 minutes at room temperature. Detection was performed using an HRP-conjugated goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:500 followed by colorimetric detection using Metal Enhanced DAB Substrate Kit (Product # 34065). Images were taken on a Zeiss Axiovision microscope at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of Ezrin using Ezrin Monoclonal Antibody (Product # MA5-13862) on Native Human Raji Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of Ezrin using Ezrin Monoclonal Antibody (Product # MA5-13862) on denatured Human Raji Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 HA is increased in circulation and in the RPE and choroid in mouse models of Sorsby''s fundus dystrophy. ( A ) HA was increased in plasma from S179C-TIMP3 knockin mice (Timp3 S179C6/S179C ) and TIMP3-KO (Timp3 -/- ) mice compared to wild-type (WT) littermates. (n >= 5). Data are presented as mean +- SEM. ( B ) Mouse retina sections stained with biotinylated HA binding protein (HABP) in the absence (upper panel) or presence (lower panel) of hyaluronidase to detect HA. HABP staining is specific for HA as shown by the absence of staining in sections treated with hyaluronidase (lower panel). Green: HA; blue: DAPI. ( C - H ) Representative images of HA staining of mouse sections from wild-type (WT) mice ( C , F ), S179C-TIMP3 mutant mice ( D , G ) and TIMP3-KO mice ( E , H ). HA is increased in the RPE and choroid of S179C-TIMP3 ( D ) and TIMP3-KO ( E ) mice compared to wild-type (WT) littermate controls ( C ). HA (green) is predominantly localized to the basal surface of the RPE (**) ( F - H ) and not to the apical surface (*) as shown by co-staining with ezrin, a marker for the apical microvilli of RPE (red). 40x images ( C - E ); 63x images ( F - H ). n >= 3 for all immunohistochemistry data.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 In epithelial cells, SIKE colocalizes with actin cytoskeletal markers. DOV 13 cells were fixed, permeabilized, and stained with mouse anti-cytoskeletal marker antibody as indicated followed by anti-mouse Alexa Fluor 555 (red), rabbit anti- SIKE antibody followed by anti-rabbit Alexa Fluor 488 (green) and Hoechst 33342 (blue). F-actin was stained with Alexa Fluor 555 Phalloidin. Cytoskeletal markers: actin, alpha-actinin, alpha-tubulin, ezrin, myosin light chain 2 ( MLC 2), focal adhesion kinase ( FAK ), beta-catenin. Images are maximum intensity of z -stack projections. Scale bar, 10 mum. Images are representative of two independent experiments, five images/experiment. Images are maximum intensity z -stack projections. Intersection of fluorescence for SIKE to target was significant (>= 0.5) for actin, alpha-actinin, and ezrin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 8 In myeloid cells, SIKE colocalizes with actin and microtubule markers. RAW 264.7 cells were fixed, permeabilized, and stained with mouse anti-cytoskeletal marker antibody as indicated followed by anti-mouse Alexa Fluor 555 (red), rabbit anti- SIKE antibody followed by anti-rabbit Alexa Fluor 488 (green) and Hoechst 33342 (blue). F-actin was stained with Alexa Fluor 555 Phalloidin. Cytoskeletal markers: actin, alpha-actinin, alpha-tubulin, ezrin, MLC 2, focal adhesion kinase ( FAK ), beta-catenin. Images are maximum intensity of z -stack projections. Scale bar, 10 mum. Images are representative of two independent experiments, five images/experiment. Images are maximum intensity z -stack projections. Intersection of fluorescence for SIKE to target was significant (>= 0.5) for alpha-actinin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 9 Quantitation of colocalization between SIKE and cytoskeletal markers. volocity software (PerkinElmer) was used to define SIKE or cytoskeletal marker volumes on thresholded images. Fraction of intersecting volumes was determined for SIKE to marker compared to total SIKE volume and marker to SIKE compared to total marker volume (A, C). These graphs represent co-occurrence. Significant co-occurrence was noted when values were > 0.5 with a 95% confidence interval. Thresholded PCC was determined (B, D) to assess codistribution of SIKE with cytoskeletal markers. Error bars represent standard error, n = 5. (A) In epithelial cells, co-occurrence of actin, alpha-actinin, and ezrin was observed for SIKE fluorescence. In myeloid cells, co-occurrence of alpha-actinin was observed for SIKE fluorescence. No significant (>= 0.5, as noted in Ref. 27 ) codistribution of cytoskeletal marker and SIKE fluorescence was observed for either epithelial (B) or myeloid (D) cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 10.1371/journal.ppat.1009716.g003 Fig 3 HAstV-1 infection leads to a disruption of cellular polarity. (A) Caco-2 infected with HAstV-1 or mock infected (as indicated). Cells were fixed at 24 hpi in 100% ice-cold methanol and then stained for ezrin (red), sodium-potassium ATPase (green), and DAPI (blue). Images are representative of at least three independent experiments. (B) Basal or apical localization of sodium-potassium ATPase was determined by measuring intensity above and below cell midline of HAstV- or mock-infected cells using ImageJ. Error bars indicate standard deviations of three independent experiments performed in triplicate, and asterisks show statistical significance as measured by the two-tailed Student t test as follows: ***, P < 0.001.