MA5-12759

antibody from Invitrogen Antibodies

Targeting: ERBB2 CD340, HER-2, HER2, NEU, NGL

Provider product page for MA5-12759

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [63]
Comments [0]
Validations
Immunocytochemistry [1]
Immunohistochemistry [2]
Other assay [5]

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Product number: MA5-12759 - Provider product page
Provider: Invitrogen Antibodies
Product name: ErbB2 (HER-2) Monoclonal Antibody (9G6.10)
Antibody type: Monoclonal
Antigen: Other
Description: MA5-12759 targets HER-2 in FACS, IF, IHC (F), and IP applications and shows reactivity with Human samples.
Antibody clone number: 9G6.10
Concentration: 0.2 mg/mL

ERBB2 protein structure - MA5-12759 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 7 Sub-cellular localization of ERbeta1-5. U2OS cells were plated on cover slips and transiently transfected with Flag-tagged expression vectors for ERbeta1-5 for 24 h. Cells were subsequently fixed, permeabilized, and incubated with either the monoclonal Flag (1:50), MC10 (1:50), or PPG5/10 (1:50) antibodies. Localization of ERbeta1-5 was determined using a FITC-labeled anti-mouse secondary antibody and confocal microscopy. A: The PPG5/10 antibody was only able to detect ERbeta1 expression while the MC10 antibody was able to detect all forms of ERbeta. ERbeta1 and ERbeta2 were completely localized within the cell nucleus while appreciable levels of cytoplasmic localization were also observed for ERbeta3-5 using both the Flag and MC10 antibodies. B: A significant number of cells transfected with ERbeta4 and ERbeta5 expression vectors also exhibited a differential sub-cellular localization pattern characterized by strong peri-nuclear and punctate cytoplasmic staining in the absence of nuclear staining with both the Flag and MC10 antibodies.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 4 Specificity and comparison of the MC10 monoclonal antibody for ERbeta protein to that of other commercial antibodies as determined by immunofluorescence. U2OS cells expressing either Flag-tagged ERbeta or ERalpha under the control of a doxycycline inducible promoter were pelleted, paraffin embedded, sectioned, and processed for immunofluorescence. Sections of un-induced cells, ERbeta expressing cells and ERalpha expressing cells were stained with a monoclonal Flag antibody (1:100) (A), an ERalpha antibody (Dako ID5, 1: 50) (B), or the following ERbeta antibodies: MC10 (1:300) (C), Chemicon (AB1410, 1:400) (D), Santa Cruz (sc-6820, 1:100) (E), Calbiochem (GR40, 1:100) (F), and PPG5/10 (1:100) (G).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 7 Sub-cellular localization of ERbeta1-5. U2OS cells were plated on cover slips and transiently transfected with Flag-tagged expression vectors for ERbeta1-5 for 24 h. Cells were subsequently fixed, permeabilized, and incubated with either the monoclonal Flag (1:50), MC10 (1:50), or PPG5/10 (1:50) antibodies. Localization of ERbeta1-5 was determined using a FITC-labeled anti-mouse secondary antibody and confocal microscopy. A: The PPG5/10 antibody was only able to detect ERbeta1 expression while the MC10 antibody was able to detect all forms of ERbeta. ERbeta1 and ERbeta2 were completely localized within the cell nucleus while appreciable levels of cytoplasmic localization were also observed for ERbeta3-5 using both the Flag and MC10 antibodies. B: A significant number of cells transfected with ERbeta4 and ERbeta5 expression vectors also exhibited a differential sub-cellular localization pattern characterized by strong peri-nuclear and punctate cytoplasmic staining in the absence of nuclear staining with both the Flag and MC10 antibodies.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 1 Relocalization of ErbB2 to the apical surface of polarized Caco-2 cells by over-expression. A: Detection of endogenous ErbB2 on untransfected permeabilized Caco-2 cells. The cells were grown on filters for 10 days and then fixed and challenged with anti-ErbB2 ECD antibody (red). B: Apical localization of transfected (over-expressed) ErbB2. The cells were grown on filters and transfected with ErbB2 4 days after seeding the culture. These confluent non-permeabilized monolayers were fixed and processed with anti-ErbB2 ECD (red channel) from the apical side of the filter. A,B: Single confocal sections in the x - y plane (top panels), and 3D reconstructions of the confocal stack in the x - z plane (perpendicular to the monolayer, apical side up, bottom panels). C: Localization of ErbB2 in polarized mock-transfected Caco-2 cells (empty vector tagged with GFP). The confluent monolayers were transfected with vector expressing GFP as described in (A and B). The fixed, non-permeabilized monolayer was processed for immunofluorescence with anti-ErbB2 ECD antibody (red) only from the apical side of the filter. Scale bars, A-C, 10 um. D: Detection of apical ErbB2 by extracellular biotinylation in ErbB2- (+) and mock-transfected cells (-). Caco-2 cells were grown on filters, transfected as described above and subjected to biotinylation from the apical or basolateral sides of the filter. Triton X-100 extracts were pulled-down with streptavidin-agarose and the SDS eluates separated by PAGE

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 2 Apical localization of ErbB2-activated tyrosine 1139 (pY1139) and phosphorylation of p38 in Caco-2 cells. A: Localization by vectorial biotinylation: Caco-2 cells were transfected with ErbB2 (+) or mock-transfected (-), biotinylated from the apical or basolateral side, and lysed. Biotinylated proteins were pulled-down with streptavidin-coupled beads. Total SDS extracts of SKOV3 cells were used as positive control for the antibody (+c). ErbB2 pY1139 and pY1248 were detected in the precipitates by SDS-PAGE and immunoblot with phospho site specific monoclonal antibodies. Total ErbB2 protein was localized in the same membranes by reprobing. B: Immunofluorescence localization of ErbB2 and its phosphorylated form, pY1139 ErbB2 by confocal microscopy in a confluent polarized Caco-2 cells. The cells were grown on filters, transfected with ErbB2 cDNA, permeabilized, and processed for immunofluorescence with anti-ErbB2 (green) and anti-ErbB2 pY1139 (red) antibodies. Confocal sections are shown in the xy plane and 3D reconstructions of the xz plane are shown apical side up (* non-transfected cell). C: Inhibition of apically localized pY1139 ErbB2 by apical Herceptin. Caco-2 cells were exposed to10 µg/mL Herceptin from the apical side for 24 h, biotinylated from the apical side, and lysed. Biotinylated proteins were pulled-down as described above, and ErbB2 pY1139 was detected by immunoblot. The control for locating the ErbB2 pY1139 band (+c) was performed as described above. Imm