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Phospho-NF-kB p65 (S536) Recombinant Rabbit Monoclonal Antibody [PSH10-58]

Usd: 385

Specification

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Catalog# HA723223

Phospho-NF-kB p65 (S536) Recombinant Rabbit Monoclonal Antibody [PSH10-58]

Application

  • WB

  • IF-Cell

Reactivity

  • Human

  • Mouse

  • Rat

BSA and Azide free
Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

Phospho-NF-kB p65 (S536) Recombinant Rabbit Monoclonal Antibody [PSH10-58]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding Ser536 of Human NF-kB p65.

Species Reactivity

Human, Mouse, Rat

Validated Applications

WB, IF-Cell

Molecular Weight

Predicted band size: 60 kDa

Positive Control

HeLa treated with 100nM Calyculin A and 20ng/mL TNF-α for 10 minutes cell lysate, NIH/3T3 treated with 100nM Calyculin A and 20ng/mL TNF-α for 10 minutes cell lysate, HeLa cells treated with 100nM Calyculin A and 20ng/mL TNF-α for 15 minutes, C6 treated with 100ng/mL Calyculin A for 1 hour cell lysate, PC-12 treated with 100nM Calyculin A for 30 minutes cell lysate.

Conjugation

unconjugated

Clone Number

PSH10-58

Reactivity Data

WB IF-Cell
Human
Tested Tested
Tested Tested
Mouse
Tested Tested
Rat
Tested Tested

Product Features

Form

Liquid

Concentration

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.

Storage Buffer

PBS (pH7.4), 0.1% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:5,000

  • IF-Cell

  • 1:500

Target

Function

Transcription factor p65 also known as nuclear factor NF-kappa-B p65 subunit is a protein that in humans is encoded by the RELA gene. RELA, also known as p65, is a REL-associated protein involved in NF-κB heterodimer formation, nuclear translocation and activation. NF-κB is an essential transcription factor complex involved in all types of cellular processes, including cellular metabolism, chemotaxis, etc. Phosphorylation and acetylation of RELA are crucial post-translational modifications required for NF-κB activation. RELA has also been shown to modulate immune responses, and activation of RELA is positively associated with multiple types of cancer.

Background References

1. Shim H et al. NF-kappaB p65 represses microRNA-124 transcription in diffuse large B-cell lymphoma. Genes Genomics. 2020 May

2. Ren C et al. Ubiquitination of NF-kappaB p65 by FBXW2 suppresses breast cancer stemness, tumorigenesis, and paclitaxel resistance. Cell Death Differ. 2022 Feb

Subcellular Location

Nucleus, Cytoplasm.

UNIPROT

SwissProt: Q04206 Human

SwissProt: Q04207 Mouse

Entrez Gene: 309165 Rat

Synonyms

Avian reticuloendotheliosis viral (v rel) oncogene homolog A antibody

MGC131774 antibody

NF kappa B p65delta3 antibody

nfkappabp65 antibody

NFkB p65 antibody

NFKB3 antibody

Nuclear factor kappaB antibody

Nuclear Factor NF Kappa B p65 Subunit antibody

Nuclear factor NF-kappa-B p65 subunit antibody

Nuclear factor of kappa light polypeptide gene enhancer in B cells 3 antibody

Expand

Images

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-NF-kB p65 (S536) on different lysates with Rabbit anti-Phospho-NF-kB p65 (S536) antibody (<a href="/products/HA723223" style="font-weight: bold;text-decoration: underline;">HA723223</a>) at 1/5,000 dilution.<br /><br />Lane 1: HeLa cell lysate (20 µg/Lane)<br />Lane 2: HeLa treated with 100nM Calyculin A and 20ng/mL TNF-α for 10 minutes cell lysate (20 µg/Lane)<br />Lane 3: NIH/3T3 cell lysate (20 µg/Lane)<br />Lane 4: NIH/3T3 treated with 100nM Calyculin A and 20ng/mL TNF-α for 10 minutes cell lysate (20 µg/Lane)<br /><br />Predicted band size: 60 kDa<br />Observed band size: 65 kDa<br /><br />Exposure time: 59 seconds; ECL: K1801;<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/HA723223" style="font-weight: bold;text-decoration: underline;">HA723223</a>) at 1/5,000 dilution was used in 5% NFDM/TBST 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-NF-kB p65 (S536) on different lysates with Rabbit anti-Phospho-NF-kB p65 (S536) antibody (HA723223) at 1/5,000 dilution.

    Lane 1: HeLa cell lysate (20 µg/Lane)
    Lane 2: HeLa treated with 100nM Calyculin A and 20ng/mL TNF-α for 10 minutes cell lysate (20 µg/Lane)
    Lane 3: NIH/3T3 cell lysate (20 µg/Lane)
    Lane 4: NIH/3T3 treated with 100nM Calyculin A and 20ng/mL TNF-α for 10 minutes cell lysate (20 µg/Lane)

    Predicted band size: 60 kDa
    Observed band size: 65 kDa

    Exposure time: 59 seconds; ECL: K1801;
    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 (HA723223) at 1/5,000 dilution was used in 5% NFDM/TBST 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 HeLa cells untreated / treated with 100nM Calyculin A and 20ng/mL TNF-α for 15 minutes labeling Phospho-NF-kB p65 (S536) with Rabbit anti-Phospho-NF-kB p65 (S536) antibody (<a href="/products/HA723223" style="font-weight: bold;text-decoration: underline;">HA723223</a>) at 1/500 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-NF-kB p65 (S536) antibody (<a href="/products/HA723223" style="font-weight: bold;text-decoration: underline;">HA723223</a>) at 1/500 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 HeLa cells untreated / treated with 100nM Calyculin A and 20ng/mL TNF-α for 15 minutes labeling Phospho-NF-kB p65 (S536) with Rabbit anti-Phospho-NF-kB p65 (S536) antibody (HA723223) at 1/500 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-NF-kB p65 (S536) antibody (HA723223) at 1/500 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.

  • Western blot analysis of Phospho-NF-kB p65 (S536) on different lysates with Rabbit anti-Phospho-NF-kB p65 (S536) antibody (<a href="/products/HA723223" style="font-weight: bold;text-decoration: underline;">HA723223</a>) at 1/5,000 dilution.<br /><br />Lane 1: C6 cell lysate (20 µg/Lane)<br />Lane 2: C6 treated with 100ng/mL Calyculin A for 1 hour cell lysate (20 µg/Lane)<br />Lane 3: PC-12 cell lysate (20 µg/Lane)<br />Lane 4: PC-12 treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane)<br /><br />Predicted band size: 60 kDa<br />Observed band size: 65 kDa<br /><br />Exposure time: 59 seconds; ECL: K1801;<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/HA723223" style="font-weight: bold;text-decoration: underline;">HA723223</a>) at 1/5,000 dilution was used in primary antibody dilution (<a href="/products/K1803" style="font-weight: bold;text-decoration: underline;">K1803</a>) 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.

    Western blot analysis of Phospho-NF-kB p65 (S536) on different lysates with Rabbit anti-Phospho-NF-kB p65 (S536) antibody (HA723223) at 1/5,000 dilution.

    Lane 1: C6 cell lysate (20 µg/Lane)
    Lane 2: C6 treated with 100ng/mL Calyculin A for 1 hour cell lysate (20 µg/Lane)
    Lane 3: PC-12 cell lysate (20 µg/Lane)
    Lane 4: PC-12 treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane)

    Predicted band size: 60 kDa
    Observed band size: 65 kDa

    Exposure time: 59 seconds; ECL: K1801;

    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 (HA723223) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Citation

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