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Purified anti-NF-κB p65 Phospho (Ser529) Antibody

Pricing & Availability
Clone
A21012B (See other available formats)
Regulatory Status
RUO
Other Names
NF-kB p65, RelA,P65, NFKB3, Nuclear Factor NF-Kappa-B P65 subunit, Transcription Factor P65, NF-Kappa-B Transcription Factor P65, RELA Proto-Oncogene NF-KB subunit
Isotype
Mouse IgG1, κ
Ave. Rating
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Product Citations
publications
1_A21012B_PURE_NF-kB_Antibody_1_042623
HeLa cells were fixed with 4% PFA Fixation Buffer (Cat. No. 420801), permeabilized with 0.5% Triton X-100 for 10 minutes, and blocked with 5% FBS for 1 hour at room temperature. The cells were either untreated (panel B), treated with TNFα without Lambda Protein Phosphatase (LPP) (panel C), or treated with TNFα and LPP (panel D), and were then incubated with 5.0 µg/mL of purified anti-NF-κB p65 Phospho (Ser529) (clone A21012B) or 5.0 μg/mL purified mouse IgG1, κ isotype control (Cat. No. 400102) (panel A) for two hours at room temperature followed by incubation with 2.5 μg/mL of Alexa Fluor® 594 goat anti-mouse IgG (Cat. No. 405326) for 1 hour at room temperature. Nuclei were counterstained with DAPI (Cat. No. 422801). Images were captured with a 60X objective. Scale bar: 50 μm
  • 1_A21012B_PURE_NF-kB_Antibody_1_042623
    HeLa cells were fixed with 4% PFA Fixation Buffer (Cat. No. 420801), permeabilized with 0.5% Triton X-100 for 10 minutes, and blocked with 5% FBS for 1 hour at room temperature. The cells were either untreated (panel B), treated with TNFα without Lambda Protein Phosphatase (LPP) (panel C), or treated with TNFα and LPP (panel D), and were then incubated with 5.0 µg/mL of purified anti-NF-κB p65 Phospho (Ser529) (clone A21012B) or 5.0 μg/mL purified mouse IgG1, κ isotype control (Cat. No. 400102) (panel A) for two hours at room temperature followed by incubation with 2.5 μg/mL of Alexa Fluor® 594 goat anti-mouse IgG (Cat. No. 405326) for 1 hour at room temperature. Nuclei were counterstained with DAPI (Cat. No. 422801). Images were captured with a 60X objective. Scale bar: 50 μm
  • 2_A21012B_PURE_NF-kB_Antibody_2_042623
    Whole cell extracts (15 µg total protein) from untreated HeLa cells (-) and HeLa cells treated with 20 ng/mL TNFα for 5 minutes (+) were resolved by 4-12% Bis-Tris gel electrophoresis, transferred to a PVDF membrane, and probed with 1.0 μg/mL purified anti-NF-κB p65 Phospho (Ser529) (clone A21012B) for 2 hours at room temperature. Proteins were visualized by chemiluminescence detection using HRP goat anti-mouse IgG (Cat. No. 405306) at a 1:3000 dilution. Equal protein loading was confirmed using a pan NF-κB p65 antibody (clone A21012A) and Direct-Blot™ HRP anti-GAPDH (Cat. No. 607904) used at a 1:50000 dilution. Western-Ready™ ECL Substrate Premium Kit (Cat. No. 426319) was used as a detection agent. Lane M: Molecular weight marker
  • 3_A21012B_PURE_NF_kB_Antibody_ICC_051123
    HeLa cells treated with 20 ng/ml TNFα and 100 nM Calyculin A for 15 minutes (filled histogram, positive control) and untreated HeLa cells (open histogram, negative control) were fixed with Fixation Buffer (Cat. No. 420801), permeabilized using True-Phos™ Perm Buffer (Cat. No. 425401), and intracellularly stained with purified anti-NF-κB p65 Phospho (Ser529) (clone A21012B) or purified mouse IgG1, κ isotype control (dashed line, open histogram) (representative histogram for untreated and treated cells) (Cat. No. 400101) followed by PE goat anti-mouse IgG (Cat. No. 405307).
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614151 25 µg 125 CHF
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614152 100 µg 385 CHF
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Description

NF-κB p65, also known as NFKB3 and RelA, is one of five members of the nuclear factor κB (NF-κB)/Rel family. Inactive NF-κB p65 is sequestered in the cytoplasm as a complex with IκB inhibitory proteins. Upon activation of the NF-κB pathway, IκB proteins are degraded, thus releasing NF-κB p65 to the nucleus. Binding of NF-κB p65 to transcriptional cofactors is induced via phosphorylation on serines 276, 529, 536, and 471. As a transcription factor, NF-κB p65 is vital to cell proliferation, immune response, survival, and apoptosis. Through regulation of chondrocyte and osteoblast differentiation and survival, NFκB p65 has a crucial role in skeletal development. Overexpressed in several cancers, NF-κB p65 functions as an oncogene and has been implicated in tumorigenesis, metastasis, tumor angiogenesis, and chemoresistance.

Product Details
Technical Data Sheet (pdf)

Product Details

Verified Reactivity
Human
Antibody Type
Monoclonal
Host Species
Mouse
Immunogen
Synthetic peptide corresponding to human NF-κB p65 phosphorylated at serine 529
Formulation
Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide
Preparation
The antibody was purified by affinity chromatography.
Concentration
0.5 mg/mL
Storage & Handling
The antibody solution should be stored undiluted between 2°C and 8°C.
Application

WB - Quality tested

ICC, ICFC - Verified
Recommended Usage

Each lot of this antibody is quality control tested by western blotting. For western blotting, the suggested use of this reagent is 0.5 - 2.0 µg/mL. For immunocytochemistry, a concentration range of 2.5 - 5.0 μg/mL is recommended. For flow cytometric staining, the suggested use of this reagent is ≤ 0.125 µg per million cells in 100 µL volume. It is recommended that the reagent be titrated for optimal performance for each application.

Application Notes

When testing by ICC, we recommend fixation with 4% PFA followed by permeabilization with Triton X-100.

RRID
AB_2941615 (BioLegend Cat. No. 614151)
AB_2941615 (BioLegend Cat. No. 614152)

Antigen Details

Structure
NF-κB p65 is a 551 amino acid protein with a predicted molecular weight of 60 kD.
Distribution

Ubiquitously expressed/ Cytoplasm and Nucleus

Function
Transcription, RAS pathway, host-virus interaction, oncogene
Interaction
NF-kappa-B, TLE5, TLE1, TP53BP2, GSK3B, MAP3K14, HDAC3, AKIP1, FOXP3
Biology Area
Cell Biology, Immuno-Oncology, Immunology, Innate Immunity, Transcription Factors
Molecular Family
Innate Immune Signaling, Phospho-Proteins, Protein Kinases/Phosphatase
Antigen References
  1. Giridharan S and Srinivasan M. 2018. J Inflamm Res. 11:407-419.
  2. Ren C, et al. 2022. Cell Death Differ. 29:381-392.
  3. Yu S, et al. 2020. FEBS Lett. 594:13681378.
Gene ID
5970 View all products for this Gene ID
UniProt
View information about NF-kappaB p65 on UniProt.org
Go To Top Version: 1    Revision Date: 04.26.2023

For Research Use Only. Not for diagnostic or therapeutic use.

 

This product is supplied subject to the terms and conditions, including the limited license, located at www.biolegend.com/terms) ("Terms") and may be used only as provided in the Terms. Without limiting the foregoing, BioLegend products may not be used for any Commercial Purpose as defined in the Terms, resold in any form, used in manufacturing, or reverse engineered, sequenced, or otherwise studied or used to learn its design or composition without express written approval of BioLegend. Regardless of the information given in this document, user is solely responsible for determining any license requirements necessary for user’s intended use and assumes all risk and liability arising from use of the product. BioLegend is not responsible for patent infringement or any other risks or liabilities whatsoever resulting from the use of its products.

 

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This data display is provided for general comparisons between formats.
Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors.
If you need assistance with selecting the best format contact our expert technical support team.

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