710768

antibody from Invitrogen Antibodies

Targeting: EGFR ERBB, ERBB1

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

710768

Provider

Invitrogen Antibodies

Product name

Phospho-EGFR (Tyr1086) Recombinant Polyclonal Antibody (11HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

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

Antibody clone number

11HCLC

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (30 µg lysate) of SH-SY5Y (lane 1), SH-SY5Y treated witrh EGF (lane 2), A549 (lane 3), A549 treated with EGF (lane 4), A-431 (lane 5), A431 treated with EGF (lane 6), Hela (lane 7) and Hela treated with EGF (lane 8) (200 ng/mL/10 min). The blots were probed with Anti-EGFR (pY1086) Recombinant Rabbit Polyclonal Antibody (Product # 710768, 0.5-2 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 130 kDa band corresponding to EGFR (pY1086) was observed across cell lines according to the given treatment. B) To confirm specificity, 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 membrane. 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 method. The membrane was probed with primary and secondary antibody following blocking with 5% nonfat 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 fixed and permeabilized A431 cells for detection of EGFR (pY1086) using Anti-EGFR (pY1086) Recombinant Rabbit Polyclonal Antibody (Product # 710768, 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) (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.