701736

antibody from Invitrogen Antibodies

Targeting: EGFR ERBB, ERBB1

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Antibody Data

Product number

701736

Provider

Invitrogen Antibodies

Product name

Phospho-EGFR (Tyr1086) Recombinant Rabbit Monoclonal Antibody (11H22L16)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Monkey, Rat, Mouse, Pig, Bovine, Rabbit and Dog.

Antibody clone number

11H22L16

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

A) Western blot analysis was performed on whole cell extracts (50 µg lysate) of A431 (Lane 1), A431 treated with EGF (200 ng/mL/ 10 minutes) (Lane 2), HeLa (Lane 3), HeLa treated with EGF (200 ng/mL/10 minutes) (Lane 4). The blots were probed with EGFR (pY1086) Recombinant Rabbit Monoclonal Antibody (Product # 701736 2-3 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 135 kDa band corresponding to EGFR (pY1086) was observed across cell lines according to the treatment. B) To confirm the specificity of Anti-EGFR (pY1086) Recombinant Rabbit Monoclonal Antibody, competition was performed with the phosphopeptide (10 µg/mL) as shown in the corresponding blot on the right. The peptide competes with the antibody and prevents it from binding to the target protein. 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 by overnight wet transfer 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).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence was performed on A431cells treated with EGF (200 ng/mL/10 minutes). These cells were permeabilized and fixed for detection of EGFR (pY1086) using Anti- EGFR (pY1086) Recombinant Rabbit Monoclonal Antibody (Product # 701736, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 0.4 µg/mL, 1:2500). Panel a) shows representative cells that were stained for detection and localization of EGFR (pY1086) protein (green), Panel b) is stained for Nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938, 1:50). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 594 Phalloidin (Product # A12381, 1:200). Panel d) is a composite image of Panels a, b and c clearly demonstrating Membrane and cytoplasmic localization of EGFR (pY1086). Panel e) represents control cells with no primary antibody to assess background.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow Cytometry analysis of EGFR [pY1086] was performed on A431 cells treated with EGF (200 ng/mL, 10 minutes) labeled with ABfinityª Anti-EGFR [pY1086] Recombinant Rabbit Monoclonal Antibody (Product# 701736, 2-4 ug/ 1M cells) or with rabbit isotype control and detected with Goat anti-Rabbit IgG (H+L) Superclonalª Secondary Antibody, Alexa Fluor¨ 488 conjugate, (Product #Ê A27034, 0.4 ug/ml, 1:2500) as represented by the red and pink histograms respectively. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control. A representative of 10,000 cells were acquired and analyzed for each sample using an Attune¨ Acoustic Focusing Cytometer (4468770).