700397

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

700397

Provider

Invitrogen Antibodies

Product name

Phospho-4EBP1 (Thr46) Recombinant Rabbit Monoclonal Antibody (34H90L46)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Rhesus monkey, mouse, rat, bovine, chicken and zebrafish based on sequence homology.

Antibody clone number

34H90L46

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 Jurkat (lane1) and HeLa (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 # 700397) 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).

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 4E-BP1 (pT46) Recombinant Rabbit Monoclonal Antibody (Product # 700397) at 2 µg-4 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 4E-BP1 (pT46). Panel e shows competition 4EBP1 (pT46) with peptide. The images were captured at 20X magnification.