- 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
-
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 -
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 -
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).
| Cat # | Size | Price | Quantity Check Availability | ||
|---|---|---|---|---|---|
| 614151 | 25 µg | $125.00 | |||
| 614152 | 100 µg | $385.00 | |||
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
- 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
-
- Giridharan S and Srinivasan M. 2018. J Inflamm Res. 11:407-419.
- Ren C, et al. 2022. Cell Death Differ. 29:381-392.
- 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
Other Formats
View All NF-kB p65 Reagents Request Custom Conjugation| Description | Clone | Applications |
|---|---|---|
| Purified anti-NF-κB p65 Phospho (Ser529) | A21012B | WB,ICC,ICFC |
| PE anti-NF-κB p65 Phospho (Ser529) | A21012B | ICFC |
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Purified anti-NF-κB p65 Phospho (Ser529)
HeLa cells were fixed with 4% PFA Fixation Buffer (Cat. No. ... 
Whole cell extracts (15 µg total protein) from untreated HeL... 
HeLa cells treated with 20 ng/ml TNFα and 100 nM Calyculin A... -
PE anti-NF-κB p65 Phospho (Ser529)
Peripheral blood mononuclear cells untreated (negative contr...