MA5-13269

antibody from Invitrogen Antibodies

Targeting: EGFR ERBB, ERBB1

Provider product page for MA5-13269

Western blot

Antibody data

Antibody Data
Antigen structure
References [71]
Comments [0]
Validations
Western blot [4]
Immunocytochemistry [3]
Immunohistochemistry [1]
Flow cytometry [1]
Other assay [4]

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Product number: MA5-13269 - Provider product page
Provider: Invitrogen Antibodies
Product name: EGFR Monoclonal Antibody (111.6)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: MA5-13269 targets Epidermal Growth Factor Receptor in IHC (P), IP, and WB applications and shows reactivity with Human samples.
Antibody clone number: 111.6
Concentration: 0.2 mg/mL

EGFR protein structure - MA5-13269 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of A-431 (Lane 1), H1975 (Lane 2), A549 (Lane 3), HeLa (Lane 4), U-87 MG (Lane 5) and MDA-MB-231 (Lane 6). The blot was probed with anti-EGFR Mouse Monoclonal Antibody (Product # MA5-13269, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 180 kDa band corresponding to EGFR was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane using the wet transfer system. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of A431 (Lane 1), Mouse Skin (lane 2) and Mouse Placenta (lane 3). The blots were probed with Anti-EGFR Mouse Monoclonal Antibody (Product # MA5-13269, 1-3 µg/mL) and detected by chemiluminescence Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 62-6520, 1:4000 dilution). Two band at 180 kDa and 60 kDa band corresponding to EGFR was observed across cell lines and tissues tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane by overnight wet transfer method. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of EGFR (Fig a) was performed by loading 30 µg of A-431 Control (lane1), A-431 EGFR knockout (lane 2) whole cell extracts using Novex® NuPAGE® 10 % Bis-Tris gel, XCell SureLock™ Electrophoresis System (EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. EGFR was detected at ~ 180 kDa using Anti-EGFR Mouse Monoclonal Antibody (Product # MA5-13269,1 µg/mL) at in 5% skim milk at 4°C overnight on a rocking platform. Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody HRP conjugate (Product # A28177, 1:4000 dilution) was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106). Densitometric analysis of this western blot is shown in histogram (Fig b). Loss of signal in CRISPR mediated knockout (KO) confirms that antibody is specific to EGFR.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-EGFR Monoclonal Antibody (111.6) (Product # MA5-13269) and a 130 kDa band corresponding to EGFR was observed across tissues and cell lines tested. Membrane enriched extracts (30 µg lysate) of Mouse Placenta (Lane 1), Mouse Brain (Lane 2), Rat Brain (Lane 3), Mouse Liver (Lane 4), Rat Liver (Lane 5), A-431 (Lane 6) were electrophoresed using NuPAGE™ 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 the primary antibody (1:500 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177,1:4000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of EGFR was performed using 90% confluent log phase A-431 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 # MA5-13269) 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: Immunofluorescence analysis of EGFR was done on 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 EGFR (111.6) Mouse Monoclonal Antibody (Product # MA5-13269) at 2 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 Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing membranous localization. Panel e is a no primary antibody control. The images were captured at 60X 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 # MA5-13269, 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of Epidermal Growth Factor Receptor using Epidermal Growth Factor Receptor Monoclonal Antibody (Product # MA5-13269) on Native Human A431 Cells.

Supportive validation

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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 8 Association between Gefitinib and OC-Exo on EGFR signaling pathway in cumulus cells. (a and b) Immunocytochemical analysis of phospho-EGFR (p-EGFR, green) and phospho-MAPK1/3 (p-MAPK1/3, green) in cumulus cells and their nuclear staining (DAPI, blue). Original magnification x100. (c) Effect of Gefitinib/OC-Exo on protein expression of p-EGFR and p-MAPK1/3 in cumulus cells. (d) Aliquots of untreated cells (control), and cells treated with 1 muM Gefitinib, with 1 muM Gefitinib and 50 ug OC-Exo, or with 50 mug OC-Exo. Cumulus cells were lysed and total cellular lysates were immunoblotted against EGFR, p-EGFR, t-MAPK1/3 (MAPK1/3), and p-MAPK1/3. Data are shown as means +- SEM. (a, b, and c) Within a column, values with different superscript letters are significantly different ( p < .05). At least three replications were performed. Control: cumulus cells cultured without Gefitinib and OC-Exo; Gefi: cumulus cells cultured with 1 muM Gefitinib; Gefi + Exo: cumulus cells cultured with 1 muM Gefitinib and 50 mug OC-Exo; Exo: cumulus cells cultured with 50 mug OC-Exo. DAPI, 4',6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; OC-Exo, oviductal exosome; MAPK, mitogen-activated protein kinase; SEM, standard error of mean [Color figure can be viewed at wileyonlinelibrary.com]

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 C. pneumoniae specifically induces externalization of phosphatidylserine (PS). a Infectivity of C. pneumoniae EBs (MOI = 10) in wild-type (WT) and PS-deficient (PSA3) CHO cells ( n = 3). mean +- s.d. Infectivity in WT CHO cells was set to 100%. Mean +- s.d. b qRT PCR analysis of internalized Cpn . Triplicate samples of EBs in CHO WT and PSA3 cells (2 hpi) were pre-treated with rCPn0473 (100 µg/mL) for 1 h prior to infection (MOI = 10) ( n = 3). Mean +- s.d. c Externalization of PS by C. pneumoniae EBs early in infection. HEp-2 cells were infected with chlamydial EBs for the indicated times (MOI = 10). Externalized PS was stained with annexin-V-FLUOS prior to fixation, followed by staining with DAPI and anti-EGFR antibody. Scale bars: 2.5 um. d Externalization of PS by the indicated chlamydial species. HEp-2 cells were infected with chlamydial EBs for 1 h (MOI = 10). Externalized PS was stained as in c . Mean (triplicates) +- s.d. ( n = 3). For all panels: *** P < 0.001, ** P < 0.01 and * P < 0.05, n.s. not significant ( P > 0.05) (Student's two-sample t test). Source data are provided as a Source Data file