710778

antibody from Invitrogen Antibodies

Targeting: IRS1 HIRS-1

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

710778

Provider

Invitrogen Antibodies

Product name

Phospho-IRS1 (Ser312) Recombinant Polyclonal Antibody (18HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Monkey, Bovine, Horse and Cat.

Antibody clone number

18HCLC

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 (30 µg lysate) of HepG2 (Lane 1), HepG2 treated with IGF1 (100 ng/mL/20 min) (Lane 2), MCF7 (Lane 3), MCF7 treated with Insulin (100 nM/10 min) (Lane 4), C2C12 (Lane 5) and C2C12 treated with Insulin (100 nM/ 10 minutes) (Lane 6).The blots were probed with Anti-IRS1 (pS312)Recombinant Rabbit Polyclonal Antibody (Product # 710778, 1-3 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 180 kDa band as a result of multiple serine phosphorylations corresponding to IRS1 (pS312) was observed across cell lines. 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 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 method. The membrane was probed with 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 MCF7 cells treated with IGF (100 ng/mL/20 min). These cells were permeabilized and fixed for detection of IRS (pS312) using Anti-IRS (pS312) Recombinant Rabbit Polyclonal Antibody (Product # 710778, 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 IRS (pS312) 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 nuclear localization of IRS (pS312). Panel e) represents cells without treatment and panel f) represents control cells with no primary antibody to assess background.