701312

antibody from Invitrogen Antibodies

Targeting: EIF4EBP1 4E-BP1, PHAS-I

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

701312

Provider

Invitrogen Antibodies

Product name

Phospho-4EBP1 (Thr46) Recombinant Rabbit Monoclonal Antibody (19H1L22)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with mouse, rat, non-human primate and rabbit based on sequence homology.

Antibody clone number

19H1L22

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of 4E-BP1 (pT46) was performed by loading 20 µg of HeLa (lane1) and HEK-293 (lane2) cell lysates using Novex®NuPAGE®4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. 4EBP1 (pT46) was detected at ~20 kDa using 4EBP1 (pT46) Recombinant Rabbit Monoclonal Antibody (Product # 701312) at 1 µg-3 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. To confirm specificity, competition was performed with phosphopeptide (10 µg/mL) as shown in the corresponding blot on right. Goat anti-Rabbit IgG-HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of Phospho-4EBP1 pThr46 in whole cell extracts from EGF-treated 293 cells using a Phospho-4EBP1 pThr46 recombinant rabbit monoclonal antibody (Product # 701312) at a dilution of 2 µg/mL. To confirm specificity, competition was performed by preincubation with the phosphopeptide to inhibit antibody binding (lane 2). Samples were detected using chemiluminescence (ECL). Results show a band at ~20kDa.

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of 4E-BP1 (pT46) was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with 4EBP1 (pT46) Recombinant Rabbit Monoclonal Antibody (Product # 701312) at 1 µg-2 µg in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Goat anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (Product # A12381). Panel d is a merged image showing nuclear localization of 4EBP1 (pT46). Panel e shows competition 4EBP1 (pT46) with peptide. The images were captured at 20X magnification.