Anti-EGFR (phospho S1046 + S1047) antibody [EP2259Y]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-EGFR (phospho S1046 + S1047) antibody [EP2259Y] (AB76300)

Immunocytochemistry/Immunofluorescence analysis of untreated and EGF (100ng/mL for 10 minutes) treated A431 cells labelling EGFR (phospho S1046 + S1047) with ab76300 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab195889, an Alexa Fluor^® 594-conjugated mouse anti-tubulin (1/200). Nuclei counterstained with DAPI (blue).

Control 1 : untreated A431 cells incubated with PBS instead of primary antibody followed by incubation with ab150077 Alexa Fluor^® 488 goat anti-rabbit IgG.
Control 2 : EGF treated A431 cells incubated with PBS instead of primary antibody followed by incubation with ab150077 Alexa Fluor^® 488 goat anti-rabbit IgG.

• IP

Lab

Immunoprecipitation - Anti-EGFR (phospho S1046 + S1047) antibody [EP2259Y] (AB76300)

Purified ab76300 at 1/40 dilution (2μg) immunoprecipitating EGFR in A431 treated with EGF whole cell lysate.
Lane 1 (input) : A431 (Human epidermoid carcinoma epithelial cell) treated with EGF whole cell lysate 10μg
Lane 2 (+) : ab76300 + A431 treated with EGF whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab76300 in A431 treated with EGF whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 180 kDa

All lanes:

Immunoprecipitation - Anti-EGFR (phospho S1046 + S1047) antibody [EP2259Y] (ab76300)

Predicted band size: 134 kDa

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• WB

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Western blot - Anti-EGFR (phospho S1046 + S1047) antibody [EP2259Y] (AB76300)

All lanes:

Western blot - Anti-EGFR (phospho S1046 + S1047) antibody [EP2259Y] (ab76300) at 1/1000000 dilution

Lane 1:

A431 cell lysate at 10 µg

Lane 2:

A431 cell lysate treated with EGF at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 134 kDa

Observed band size: 180 kDa

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• Dot

Unknown

Dot Blot - Anti-EGFR (phospho S1046 + S1047) antibody [EP2259Y] (AB76300)

Primary antibody : ab76300 at a dilution of 1/1000.

Secondary antibody : Peroxidase conjugated-goat anti-rabbit IgG, (H+L) at a dilution of 1/2500.

Blocking and dilution buffer : 5% NFDM/TBST

Lane 1 : EGFR (pS1046/pS1047) phospho peptide.
Lane 2 : EGFR non-phospho peptide.

Exposure time : 3 minutes.