Anti-EGFRvIII antibody [EPR28380-83]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFRvIII antibody [EPR28380-83] (AB313646)

Immunohistochemical analysis of formalin fixed paraffin-embedded human glioblastoma and placenta tissues labelling EGFRvIII with ab313646 and EGFR with ab52894 at a concentration of 2.5 µg/ml and 0.5 µg/ml.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins. Primary antibodies were incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin.

Tissue obtained from the Human Research Tissue Bank, supported by the NHR Cambridge Biomedical Research Centre.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-EGFRvIII antibody [EPR28380-83] (AB313646)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T (human embryonic kidney epithelial cell) labelling EGFRvIII with ab313646 at 1/500 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Cells were counterstained with anti-Myc tag Mouse mab [9E10] (Alexa Fluor^® 594) at 1/100 dilution (Red). The Nuclear counterstain is DAPI (Blue).

Confocal image showing cytoplasmic and membrane staining in 293T cells overexpressing human EGFRvIII. No staining in 293T cells overexpressing human wild-type EGFR. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 (2 µg/mL) dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFRvIII antibody [EPR28380-83] (AB313646)

Chromogenic duplex (2-plex) immunohistochemical staining of formalin fixed paraffin-embedded human glioblastoma tissue performed on a Roche Ventana Discovery Ultra instrument.

Heat mediated antigen retrieval was performed with CC1 solution for 64 min at 95°C. Two rounds of primary antibody incubation were performed each for 16 min at 37°C. anti-EGFRvIII (ab313646; 1st primary antibody; 3 µg/ml) and anti-EGFR (ab52894; 2nd primary antibody; 0.5 µg/ml). Antibody denaturation was conducted after the first round of staining using Ultra CC2 solution for 8 min at 100°C to avoid cross reactivity.

Signal was developed with anti-rabbit HQ followed by anti-HQ HRP coupled with purple (Discovery purple kit (RUO) #760-229) and teal (Discovery teal kit (RUO) #760-247) chromogens and counterstained with haematoxylin II.

Tissue obtained from the Human Research Tissue Bank, supported by the NHR Cambridge Biomedical Research Centre.

• Flow Cyt

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Flow Cytometry - Anti-EGFRvIII antibody [EPR28380-83] (AB313646)

Flow cytometric analysis of 293T (human embryonic kidney epithelial cell) transfected with a human EGFRvIII expression vector containing a His-Myc tag (top left, bottom left); human wild-type EGFR expression vector containing a His-Myc tag (top right); empty vector containing a His-Myc tag (bottom right). Cells were fixed with 2% paraformaldehyde and permeabilised with 0.1% Tween-20. Cell labelling of EGFRvIII with ab313646 at 1/500 dilution. A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) was used at 1/5000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730). No cross-reactivity with human wild-type EGFR.

Cells were surface stained with primary antibody. Fixed with 2% PFA for 10 min followed by intracellularly staining with anti-Myc tag conjugated to Alexa Fluor^® 647.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFRvIII antibody [EPR28380-83] (AB313646)

Immunohistochemical analysis of paraffin-embedded 293T (human embryonic kidney epithelial cell) transfected with (A) human EGFRvIII expression vector containing a Myc-His tag; (B) human wild-type EGFR expression vector containing a Myc-His tag; (C) empty vector containing a His tag. Whole cell pellet labelling of EGFRvIII with ab313646 at 1/100 (5.09 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on 293T overexpressing human EGFRvIII (image A). No staining on 293T overexpressing human wild-type EGFR (image B) or the empty vector control (image C). The section was incubated with ab313646 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFRvIII antibody [EPR28380-83] (AB313646)

Immunohistochemical analysis of paraffin-embedded (A, D) human placenta; (B, E) human glioblastoma; (C, F) human lung adenocarcinoma tissue.

A-C : Sections were incubated with anti-EGFR antibody [SP84] - C-terminal (ab227642; 1 : 100 ) for 30 mins at room temperature. Positive staining on human placenta (image A), glioblastoma (image B), and lung adenocarcinoma (image C). D-F : Sections were incubated with anti-EGFRvIII antibody [EPR28380-83] (ab313646; 1 : 100 [5.09 µg/mL]) for 30 mins at room temperature. Positive staining on human glioblastoma (image E), no staining on placenta (image D) or lung adenocarcinoma (image F). The immunostaining was performed on a Leica Biosystems BOND® RX instrument counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IP

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Immunoprecipitation - Anti-EGFRvIII antibody [EPR28380-83] (AB313646)

EGFRvIII was immunoprecipitated from 0.35 mg of 293T (human embryonic kidney epithelial cell) transfected with a human EGFRvIII expression vector containing a His-tag using ab313646 at 1/30 dilution (2µg in 0.35mg whole cell lysate). Western blot was performed on the immunoprecipitate using ab313646 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP)(ab131366) was used at 1/5000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730).

Lanes 1-3 : 293T (human embryonic kidney epithelial cell) transfected with a human EGFRvIII expression vector containing a His-tag

Lane 1 : whole cell lysate at 0.3µg

Lane 2 : IP with ab313646

Lane 3 : IP with Isotype control (ab172730)

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-EGFRvIII antibody [EPR28380-83] (ab313646) at 1/30 dilution

All lanes:

293T (human embryonic kidney epithelial cell) transfected with a human EGFRvIII expression vector containing a His-tag whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

false

Exposure time: 5s

• WB

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Western blot - Anti-EGFRvIII antibody [EPR28380-83] (AB313646)

Blocking and diluting buffer and concentration : 5% NFDM/TBST This antibody recognizes human EGFRvIII but not human wild-type EGFR. In western blot, anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution, anti-His antibody (ab213204) staining at 1/5000 dilution, and anti-EGFR antibody staining at 1/1000 dilution.

All lanes:

Western blot - Anti-EGFRvIII antibody [EPR28380-83] (ab313646) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with an empty vector containing a His tag, whole cell lysate at 10 µg

Lane 2:

293T transfected with a human EGFR expression vector containing a His tag, whole cell lysate at 10 µg

Lane 3:

293T transfected with a human EGFRvIII expression vector containing a His tag, whole cell lysate at 10 µg

Lane 4:

293T transfected with a human EGFRvIII expression vector containing a His tag, whole cell lysate at 2.5 µg

Lane 5:

293T transfected with a human EGFRvIII expression vector containing a His tag, whole cell lysate at 0.62 µg

Lane 6:

293T transfected with a human EGFRvIII expression vector containing a His tag, whole cell lysate at 0.31 µg

Lane 7:

293T transfected with a human EGFRvIII expression vector containing a His tag, whole cell lysate at 0.16 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 134 kDa

false

Exposure time: 5.5s

• WB

Supplier Data

Western blot - Anti-EGFRvIII antibody [EPR28380-83] (AB313646)

Blocking and diluting buffer and concentration : 5% NFDM/TBST This antibody does not recognize human wild-type EGFR. (A) : anti-EGFRvIII antibody (ab313646) staining at 1/1000 dilution, (B) : anti-EGFR antibody staining at 1/1000 dilution, (C) : anti-GAPDH antibody [EPR16891] – Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-EGFRvIII antibody [EPR28380-83] (ab313646) at 1/1000 dilution

Lane 1:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

EGFR knockout HeLa whole cell lysate at 20 µg

Lane 4:

Human placenta tissue lysate at 20 µg

Secondary

Lanes 1 - 3:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Lane 4:

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

Observed band size: 134 kDa

false

Exposure time: 180s