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Invitrogen
Phospho-IkB alpha (Ser32, Ser36) Monoclonal Antibody (RILYB3R), eFluor™ 660, eBioscience™
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FIGURE: 1 / 2
Phospho-IkB alpha (Ser32, Ser36) Antibody (50-9035-41) in Flow
Intracellular staining of untreated (orange histogram) or 60-minute Human TNF alpha Recombinant Protein (Product # 14-8329-81) and Calyculin A-treated (purple histogram) normal human peripheral blood cells with Anti-Human/Mouse phospho-IkBa (S32/S36) eFluor® 660. Cells in the lymphocyte gate were used for analysis. Cells were stained using the Intracellular Fixation & Permeabilization Buffer Set (Product # ... View More
Product Details
50-9035-41
Applications
Tested Dilution
Publications
Product Specifications
Species Reactivity
Human,
Mouse
Published species
Human,
Mouse
Host/Isotype
Mouse
/ IgG2b, kappa
Recommended Isotype Control
Class
Monoclonal
Type
Antibody
Clone
RILYB3R
Conjugate
Excitation/Emission Max
651/668 nm
View spectra
Form
Liquid
Concentration
5 µL/Test
Purification
Affinity chromatography
Storage buffer
PBS, pH 7.2, with BSA
Contains
0.09% sodium azide
Storage conditions
4°C, store in dark, DO NOT FREEZE!
Shipping conditions
Ambient (domestic); Wet ice (international)
RRID
Product Specific Information
Description: This RILYB3R monoclonal antibody recognizes human and mouse nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (I kappa B alpha) when phosphorylated on serines 32 and 36 (S32/S36). I kappa B alpha is one member of a family of cellular proteins that functions to inhibit classical/canonical NF-kappa B signaling by masking the nuclear localization signals (NLS) of NF-kappa B proteins and keeping them sequestered in an inactive state in the cytoplasm. Classical/canonical NF-kappa B signaling is initiated in response to myriad stimuli including engagement of T cell and B cell receptors, growth factors, and inflammatory stimuli (reactive oxygen species, TNF alpha, IL-1) and results in the activation of the I kappa B kinase (IKK) complex that includes IKK alpha, IKK beta, and NEMO. IKK phosphorylates I kappa B alpha resulting in its ubiquitination, degradation, and subsequent translocation of NF-kappa B transcription factor proteins into the nucleus.
Applications Reported:This RILYB3R antibody has been reported for use in intracellular staining followed by flow cytometric analysis.
Applications Tested: This RILYB3R 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 (0.125 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.
Staining Protocol: Protocol A and Protocol C are recommended for this monoclonal antibody. Use of Protocol A: Two-step protocol: intracellular (cytoplasmic) proteins allows for the greatest flexibility for detection of surface and intracellular (cytoplasmic) proteins. Protocol C: Two-step protocol: Fixation/Methanol allows for the greatest discrimination of phospho-specific signaling between unstimulated and stimulated samples, but with limitations on the ability to stain specific surface proteins (refer to "Clone Performance Following Fixation/Permeabilization" located in the BestProtocols Section under the Resources tab online). All Protocols can be found in the Flow Cytometry Protocols: "Staining Intracellular Antigens for Flow Cytometry Protocol" located in the BestProtocols® Section under the Resources tab online.
eFluor® 660 is a replacement for Alexa Fluor® 647. eFluor® 660 emits at 659 nm and is excited with the red laser (633 nm). Please make sure that your instrument is capable of detecting this fluorochrome.
Excitation: 633-647 nm; Emission: 668 nm; Laser: Red Laser.
Filtration: 0.2 µm post-manufacturing filtered.
Target Information
IkB-alpha is a 40 kDa protein that functions to inhibit NF- kappaB activity. The inhibition occurs via protein-protein interaction between I kappaB proteins and NF- kappaB dimers in the cytosol. The interaction of I kappa B-alpha with NF- kappaB masks the nuclear localization sequence of NF- kappaB, preventing NF- kappaB translocation to the nucleus. A variety of stimuli can activate gene expression by liberating NF- kappaB through the degradation of I kappaB alpha. These stimuli include the proinflammatory cytokines TNF- alpha and IL-1 beta, chemokines, PMA, growth factors, LPS, UV irradiation, viral infection, as well as various chemical and physical stresses. In humans, the gene is located on the q arm of chromosome 14. Activation of NFkB requires that IkB be phosphorylated on specific serine residues, which results in targeted degradation of IkB. IkB kinase alpha (IKK alpha), previously designated CHUK, interacts with IkB-alpha and specifically phosphorylates IkB-alpha on the sites that trigger its degradation Serines 32 and 36. IKK alpha appears to be critical for NFkB activation in response to proinflammatory cytokines. Phosphorylation of IkB by IKK alpha is stimulated by the NFkB inducing kinase (NIK), which itself is a central regulator for NFkB activation in response to TNF and IL-1. The functional IKK complex contains three subunits, IKK alpha, IKK beta and IKK gamma, and each appear to make essential contributions to IkB phosphorylation.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
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Bioinformatics
Protein Aliases: I(Kappa)B(alpha); I-kappa-B-alpha; I-kappaBalpha; IkappaBalpha; IkB-alpha; IKBalpha; Major histocompatibility complex enhancer-binding protein MAD3; NF-kappa-B inhibitor alpha; nuclear factor of kappa light chain gene enhancer in B-cells; nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha; nuclear factor of kappa light polyp gene enhancer in B-cell 1; nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha
Gene Aliases: AI462015; IKBA; MAD-3; MAD3; NFKBI; NFKBIA
UniProt ID: (Human) P25963, (Mouse) Q9Z1E3
Entrez Gene ID: (Human) 4792, (Mouse) 18035
protein import into nucleus, translocation
stimulatory C-type lectin receptor signaling pathway
apoptotic process
cytoplasmic sequestering of NF-kappaB
negative regulation of macrophage derived foam cell differentiation
positive regulation of cholesterol efflux
negative regulation of lipid storage
viral process
lipopolysaccharide-mediated signaling pathway
negative regulation of NF-kappaB transcription factor activity
positive regulation of cellular protein metabolic process
response to muramyl dipeptide
toll-like receptor 4 signaling pathway
response to muscle stretch
Fc-epsilon receptor signaling pathway
regulation of cell proliferation
regulation of NF-kappaB import into nucleus
cytoplasmic sequestering of transcription factor
negative regulation of apoptotic process
response to exogenous dsRNA
negative regulation of DNA binding
negative regulation of myeloid cell differentiation
negative regulation of Notch signaling pathway
positive regulation of transcription from RNA polymerase II promoter
T cell receptor signaling pathway
positive regulation of NF-kappaB transcription factor activity
cellular response to cold
nucleotide-binding oligomerization domain containing 1 signaling pathway
nucleotide-binding oligomerization domain containing 2 signaling pathway
cellular response to cytokine stimulus
cellular response to organic cyclic compound
regulation of gene expression
response to lipopolysaccharide
positive regulation of transcription, DNA-templated
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