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Phospho-NFkB p65 (Ser529) Monoclonal Antibody (B33B4WP), PerCP-eFluor™ 710, eBioscience™
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FIGURE: 1 / 3
Phospho-NFkB p65 (Ser529) Antibody (46-9863-41) in Flow
Intracellular staining of unstimulated (left) or 15-minute PMA-stimulated (right) normal human peripheral blood cells with Anti-Human CD57 FITC (Product # 11-0577-42) and Anti-Human phospho-NF kappa B p65 (S529) PerCP-eFluor® 710, using the Intracellular Fixation and Permeabilization Buffer Set (Product # 88-8824-00) and protocol. Cells in the lymphocyte gate were used for analysis.
Product Details
46-9863-41
Applications
Tested Dilution
Publications
Product Specifications
Published species
Mouse
Host/Isotype
Mouse
/ IgG2a, kappa
Recommended Isotype Control
Class
Monoclonal
Type
Antibody
Clone
B33B4WP
Conjugate
Excitation/Emission Max
482/708 nm
View spectra
Form
Liquid
Concentration
5 µL/Test
Storage conditions
4°C, store in dark, DO NOT FREEZE!
Shipping conditions
Wet ice
RRID
Product Specific Information
Description: The B33B4WP monoclonal antibody recognizes human NF kappa B (NFkB) p65 subunit when serine 529 is phosphorylated. NFkB, also known as nuclear factor kappa-light chain enhancer of activated B cells, is a ubiquitous transcription factor that regulates the transcription of many genes involved in cell proliferation, apoptosis, development, immunity and cancer. Functional NFkB is a homo- or hetero-dimer composed of 5 members of the NFkB family: p65 (RelA), c-Rel, RelB, p50 (NFkB1, p105 precursor protein), and p52 (NFkB2, p100 precursor protein). The activity of the complex is negatively regulated by binding to IkB inhibitors that sequester NFkB into the cytoplasm, inhibiting its transcriptional activity. NFkB-activating agents like tumor necrosis factor (TNF) alpha, interleukin-1 beta, lipopolysaccharide, camptothecin, and phorbol ester (PMA) induce the phosphorylation and degradation of IkB, leading to the translocation of NFkB to the nucleus where it binds to kB motifs and regulates gene expression. The activity of p65-containing NFkB complexes is positively regulated by phosphorylation of the p65 subunit at serine 529 and/or serine 536.
The B33B4WP monoclonal antibody recognizes a single band at 65 kDa in reduced SDS lysates from TNF alpha-stimulated HeLa cells.
Applications Reported: This B33B4WP antibody has been reported for use in intracellular staining followed by flow cytometric analysis.
Applications Tested: This B33B4WP antibody has been pre-titrated and tested by intracellular staining followed by flow cytometric analysis of normal human peripheral blood cells. This can be used at 5 µL (0.03 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test.
PerCP-eFluor® 710 emits at 710 nm and is excited with the blue laser (488 nm); it can be used in place of PerCP-Cyanine5.5. We recommend using a 710/50 bandpass filter, however, the 695/40 bandpass filter is an acceptable alternative. Please make sure that your instrument is capable of detecting this fluorochrome.
Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light.
Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.
Protocols: We recommend Protocol A: Two-step protocol: intracellular (cytoplasmic) proteins. Alternatively, Protocol C: Two-step protocol: Fixation/Methanol can also be used. Protocol B: One-step protocol: intracellular (nuclear) proteins cannot be used. All Protocols can be found in the "Staining intracellular Antigens for Flow Cytometry Protocol" located in the BestProtocols® Section under the Resources tab online.
Excitation: 488 nm; Emission: 710 nm; Laser: Blue Laser.
Filtration: 0.2 µm post-manufacturing filtered.
Target Information
Nuclear factor kB p65 (NF-kB p65) is encoded by the RELA gene and is present on chromosome 11 in humans. NF-kB P65 is also known as RelA (v-rel avian reticuloendotheliosis viral oncogene homolog A) and belongs to the Rel family of proteins. It is one of the two subunits of NF-kB (Nuclear factor kappa-light-chain-enhancer of activated B cells) that heterodimerizes with the other subunits p50 or p52. However, binding of TNF alpha to its cognate receptor phosphorylates IKK which in turn phosphorylates I kB allowing proteasomal degradation of I kB. It specifically plays a key role in transcription of immunoglobulin k (kappa) gene in mature B-lymphoid cells.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
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