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Phospho-eIF4EBP1 (S65) Recombinant Rabbit Monoclonal Antibody [PSH09-44] - BSA and Azide free

Usd: 649

Specification

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Catalog# HA751291

Phospho-eIF4EBP1 (S65) Recombinant Rabbit Monoclonal Antibody [PSH09-44] - BSA and Azide free

Application

  • WB

  • IF-Cell

  • IP

Reactivity

  • Human

With BSA and Azide
Conjugation

  • unconjugated

Overview

Product Name

Phospho-eIF4EBP1 (S65) Recombinant Rabbit Monoclonal Antibody [PSH09-44] - BSA and Azide free

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding Ser65 of human eIF4EBP1.

Species Reactivity

Human

Validated Applications

WB, IF-Cell, IP

Target Molecular Weight

Predicted band size: 13 kDa

Positive Control

293T cells starved for 24 hours then treated with 100nM insulin for 30 minutes.

Conjugation

unconjugated

Clone Number

PSH09-44

Product Features

Form

Liquid

Concentration

1mg/ml

Storage Instructions

Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.

Storage Buffer

1*PBS (pH7.4).

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:1,000

  • IF-Cell

  • 1:1,000

  • IP

  • 1-2μg/sample

Target

Function

This gene encodes one member of a family of translation repressor proteins. The protein directly interacts with eukaryotic translation initiation factor 4E (eIF4E), which is a limiting component of the multisubunit complex that recruits 40S ribosomal subunits to the 5' end of mRNAs. Interaction of this protein with eIF4E inhibits complex assembly and represses translation. This protein is phosphorylated in response to various signals including UV irradiation and insulin signaling, resulting in its dissociation from eIF4E and activation of cap-dependent mRNA translation.

Background References

1. Voeltzke K et al. EIF4EBP1 is transcriptionally upregulated by MYCN and associates with poor prognosis in neuroblastoma. Cell Death Discov. 2022 Apr

2. Nelson ED et al. High EIF4EBP1 expression reflects mTOR pathway activity and cancer cell proliferation and is a biomarker for poor breast cancer prognosis. Am J Cancer Res. 2024 Jan

Subcellular Location

Cytoplasm, cytosol.

UNIPROT

SwissProt: Q13541 Human

Synonyms

4E-BP1 antibody

4EBP1 antibody

4EBP1_HUMAN antibody

BP 1 antibody

eIF4E binding protein 1 antibody

eIF4E-binding protein 1 antibody

Eif4ebp1 antibody

Eukaryotic translation initiation factor 4E-binding protein 1 antibody

PHAS-I antibody

PHASI antibody

Expand

Images

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-eIF4EBP1 (S65) on different lysates with Rabbit anti-Phospho-eIF4EBP1 (S65) antibody (<a href="/products/HA751291" style="font-weight: bold;text-decoration: underline;">HA751291</a>) at 1/1,000 dilution.<br /><br />Lane 1: 293T starved for 24 hours cell lysate<br />Lane 2: 293T starved for 24 hours then treated with 100nM insulin for 30 minutes cell lysate<br />Lane 3: 293T starved for 24 hours then treated with 100nM insulin for 30 minutes cell lysate, then the membrane treated with λpp for 1 hour<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 13 kDa<br />Observed band size: 20 kDa<br /><br />Exposure time: 3 minutes; ECL: K1802;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/HA751291" style="font-weight: bold;text-decoration: underline;">HA751291</a>) at 1/1,000 dilution was used in primary antibody dilution (WB500D) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    ☑ Cell treatment (CT)

    Western blot analysis of Phospho-eIF4EBP1 (S65) on different lysates with Rabbit anti-Phospho-eIF4EBP1 (S65) antibody (HA751291) at 1/1,000 dilution.

    Lane 1: 293T starved for 24 hours cell lysate
    Lane 2: 293T starved for 24 hours then treated with 100nM insulin for 30 minutes cell lysate
    Lane 3: 293T starved for 24 hours then treated with 100nM insulin for 30 minutes cell lysate, then the membrane treated with λpp for 1 hour

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 13 kDa
    Observed band size: 20 kDa

    Exposure time: 3 minutes; ECL: K1802;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA751291) at 1/1,000 dilution was used in primary antibody dilution (WB500D) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Immunocytochemistry analysis of 293T cells starved for 24 hours then treated with 100nM insulin for 30 minutes labeling Phospho-eIF4EBP1 (S65) with Rabbit anti-Phospho-eIF4EBP1 (S65) antibody (<a href="/products/HA751291" style="font-weight: bold;text-decoration: underline;">HA751291</a>) at 1/1,000 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-eIF4EBP1 (S65) antibody (<a href="/products/HA751291" style="font-weight: bold;text-decoration: underline;">HA751291</a>) at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />Beta tubulin (<a href="/products/HA601187" style="font-weight: bold;text-decoration: underline;">HA601187</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    ☑ Cell treatment (CT)

    Immunocytochemistry analysis of 293T cells starved for 24 hours then treated with 100nM insulin for 30 minutes labeling Phospho-eIF4EBP1 (S65) with Rabbit anti-Phospho-eIF4EBP1 (S65) antibody (HA751291) at 1/1,000 dilution.

    Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-eIF4EBP1 (S65) antibody (HA751291) at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

  • Phospho-eIF4EBP1 (S65) was immunoprecipitated from 0.2 mg 293T cell lysate with <a href="/products/HA751291" style="font-weight: bold;text-decoration: underline;">HA751291</a> at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using <a href="/products/HA751291" style="font-weight: bold;text-decoration: underline;">HA751291</a> at 1/1,000 dilution. Alpaca anti-Rabbit IgG Fc secondary antibody (<a href="/products/HA1031" style="font-weight: bold;text-decoration: underline;">HA1031</a>) at 1/50,000 dilution was used for 1 hour at room temperature.<br /><br />Lane 1: 293T cell lysate (input)<br />Lane 2: <a href="/products/HA751291" style="font-weight: bold;text-decoration: underline;">HA751291</a> IP in 293T cell lysate<br />Lane 3: Rabbit IgG instead of <a href="/products/HA751291" style="font-weight: bold;text-decoration: underline;">HA751291</a> in 293T cell lysate<br /><br />Blocking/Dilution buffer: 5% NFDM/TBST<br />Exposure time: 18 seconds; ECL: <a href="/products/K1801" style="font-weight: bold;text-decoration: underline;">K1801</a>

    Phospho-eIF4EBP1 (S65) was immunoprecipitated from 0.2 mg 293T cell lysate with HA751291 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA751291 at 1/1,000 dilution. Alpaca anti-Rabbit IgG Fc secondary antibody (HA1031) at 1/50,000 dilution was used for 1 hour at room temperature.

    Lane 1: 293T cell lysate (input)
    Lane 2: HA751291 IP in 293T cell lysate
    Lane 3: Rabbit IgG instead of HA751291 in 293T cell lysate

    Blocking/Dilution buffer: 5% NFDM/TBST
    Exposure time: 18 seconds; ECL: K1801

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