MA5-12880

antibody from Invitrogen Antibodies

Targeting: EGFR ERBB, ERBB1

Antibody data

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Validation data

Product number: MA5-12880 - Provider product page
Provider: Invitrogen Antibodies
Product name: EGFR Monoclonal Antibody (225)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA5-12880 targets Epidermal Growth Factor Receptor in IP and IF applications and shows reactivity with Human and Non-human primate samples.
Antibody clone number: 225
Concentration: 0.2 mg/mL

EGFR protein structure - MA5-12880 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of EGFR was performed using 90% confluent log phase A431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with EGFR Mouse monoclonal antibody (Product # MA5-12880) at 5 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing membrane localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of EGFR was achieved by transfecting A-431 cells with EGFR specific siRNA (Silencer® select Cat # s563, s564, s565). Immunofluorescence analysis was performed on A431 cells (untransfected, panel a,d), transfected with non-specific scrambled siRNA (panels b,e) and transfected with EGFR specific siRNA (panel c,f) Cells were fixed, permeabilized, and labelled with EGFR Mouse monoclonal Antibody (Product # MA5-12880, 5 µg/mL), followed by Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Loss of signal was observed upon siRNA mediated knockdown (panel c,f) confirming specificity of the antibody to EGFR(green). The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Phalloidin (blue) and EGFR (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 in PBS + 0.1% Triton X-100 (Product # 37525) for 30 minutes at room temperature. Cells were probed with an EGFR monoclonal antibody (Product # MA5-12880) 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 350 Phalloidin (Product # 21830) at a dilution of 1:120 (2.5units/mL final concentration) and nuclei (red) were stained with DRAQ5 (Product # 62254) 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 (orange) and EGFR (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 EGFR monoclonal antibody (Product # MA5-12880) 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 (purple) and EGFR (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 EGFR monoclonal antibody (Product # MA5-12880) 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 650 Phalloidin (Product # 21838) 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: Immunofluorescence analysis of EGFR was performed using 70% confluent log phase A-431 cells (WIld type, panels a,d), CAS9 control (panels b,e) and EGFR Knockout (panels c,f). The cells were fixed, permeabilized, and labelled with EGFR Mouse Monoclonal Antibody(Product # MA5-12880, 5 µg/mL), followed by Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175, 1:2000). Nuclei (blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938) and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Loss of signal was observed in EGFR Knockout cells (panel c,f) confirming specificity of the antibody to EGFR(green). The images were captured at 60X magnification.