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PE Mouse anti-Human IKKγ (pS376)
PE Mouse anti-Human IKKγ (pS376)

Analyses of IKKγ (pS376) expression.

   Panel 1: Multicolor flow cytometric analysis of IKKγ (pS376) expression by human T lymphocytes. Human whole blood (collected with heparin) was either left unstimulated (dashed line histogram) or stimulated (solid line histogram) with 400 nM PMA and 250 ng/ml Ionomycin (Sigma, Cat No. I-0634) at 37°C for 10 minutes. Cells were fixed in 1X BD Phosflow Lyse/Fix Buffer (Cat. No. 558049 for 5X stock) at 37°C for 10 min. The cells were permeabilized in BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for at least 30 min (or overnight at -20°C), washed and then stained with BD Phosflow™ PE Mouse anti-Human IKKγ (pS376) (Cat. No. 562590) and APC Mouse Anti-Human CD3 (Cat. No. 561810/555335/561811) antibodies. The fluorescence histograms were derived from CD3 positive-gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry was performed using a BD FACSCanto™ II Flow Cytometer System.

   Panel 2: Western blot analysis of IKKγ (pS376) in human epithelioid carcinoma. HeLa S3 cells (ATCC CCL 2.2) were cultured overnight in medium containing 0.1% FBS and then either not treated (C) or stimulated (T) with recombinant human Tumor Necrosis Factor (Cat. No. 554618; 25 ng/ml) and Calyculin A (Calbiochem Cat. No. 208851; 50 nM) at 37°C for 10 minutes. Lysates (15 µg total cell protein/lane) were blotted using Purified Mouse Anti-Human IKKγ (pS376) antibody (Cat. No. 562589; at 1.0, 0.5 and 0.25 µg/ml for Lanes 1, 2, 3, respectively), HRP Goat Anti-Mouse Ig (Cat. No. 554002) and a chemiluminescent detection system. Phosphorylated IKKγ proteins were identified as ~50-55 kDa bands by blotting.

   Panel 3: Western blot analysis of IKKγ (pS376) expressed by human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated with 50 nM Phorbol 12-Myristate 13-Acetate (PMA, Sigma, Cat. No. P-8139) at 37°C for 10 minutes, and lysates were analyzed by Western blotting as described above.

Analyses of IKKγ (pS376) expression.

   Panel 1: Multicolor flow cytometric analysis of IKKγ (pS376) expression by human T lymphocytes. Human whole blood (collected with heparin) was either left unstimulated (dashed line histogram) or stimulated (solid line histogram) with 400 nM PMA and 250 ng/ml Ionomycin (Sigma, Cat No. I-0634) at 37°C for 10 minutes. Cells were fixed in 1X BD Phosflow Lyse/Fix Buffer (Cat. No. 558049 for 5X stock) at 37°C for 10 min. The cells were permeabilized in BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for at least 30 min (or overnight at -20°C), washed and then stained with BD Phosflow™ PE Mouse anti-Human IKKγ (pS376) (Cat. No. 562590) and APC Mouse Anti-Human CD3 (Cat. No. 561810/555335/561811) antibodies. The fluorescence histograms were derived from CD3 positive-gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry was performed using a BD FACSCanto™ II Flow Cytometer System.

   Panel 2: Western blot analysis of IKKγ (pS376) in human epithelioid carcinoma. HeLa S3 cells (ATCC CCL 2.2) were cultured overnight in medium containing 0.1% FBS and then either not treated (C) or stimulated (T) with recombinant human Tumor Necrosis Factor (Cat. No. 554618; 25 ng/ml) and Calyculin A (Calbiochem Cat. No. 208851; 50 nM) at 37°C for 10 minutes. Lysates (15 µg total cell protein/lane) were blotted using Purified Mouse Anti-Human IKKγ (pS376) antibody (Cat. No. 562589; at 1.0, 0.5 and 0.25 µg/ml for Lanes 1, 2, 3, respectively), HRP Goat Anti-Mouse Ig (Cat. No. 554002) and a chemiluminescent detection system. Phosphorylated IKKγ proteins were identified as ~50-55 kDa bands by blotting.

   Panel 3: Western blot analysis of IKKγ (pS376) expressed by human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated with 50 nM Phorbol 12-Myristate 13-Acetate (PMA, Sigma, Cat. No. P-8139) at 37°C for 10 minutes, and lysates were analyzed by Western blotting as described above.

Product Details
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BD Phosflow™
IKBKG; IKKγ; IKKG; IKK-gamma; AMCBX1; FIP3; IKKAP1; IP1; IP2; IPD2; NEMO
Human (QC Testing)
Mouse IgG1, κ
Phosphorylated Human IKKγ Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_11154421
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed.

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  2. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  3. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
562590 Rev. 1
Antibody Details
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N19-39

The N19-39 monoclonal antibody specifically binds to the IKK-gamma protein phosphorylated at the Ser376 site, ie,  IKKγ (pS376). IKKγ is also known an NEMO (NF-kappa-B essential modifier) and is encoded by the IKBKG (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma) gene. The nuclear transcription factor kappa-B, NF-κB, is controlled by interaction with an inhibitory subunit, IκB, which restricts NF-κB to the cytoplasm. Following stimulation by various cytokines or other stimuli, IκB becomes degraded and NF-κB is released to the nucleus. The release of IκB from NF-κB is thought to be a critical point in the activation of NF-κB signal pathways. Activated NF-κB regulates genes involved in various pathways including inflammation, immunity, and cell survival signaling pathways. A group of proteins form an NF-κB regulatory complex, or signalsome. Two members of this complex are a pair of closely related serine/threonine kinases, IKKα and IKKβ (also called IKK-1 and IKK-2), which phosphorylate critical residues of IκB, thus targeting it for subsequent degradation. The IKK complex contains similar amounts of IKKα, IKKβ as well as two other polypeptides, which are differentially processed forms of a third subunit, IKKγ. IKKα and IKKβ become activated following phosphorylation by upstream kinases, including NF-κB-inducing kinase (NIK)4 and MEKK1. IKKβ has been shown to phosphorylate IKKγ at serine 376 in response to signaling through the TNF receptor or the Tax oncoprotein of human T-cell leukemia virus type 1. IKKγ has an essential regulatory role in the IKK complex and is required for the activation of the NF-κB pathway in response to multiple cellular stimuli. IKKγ isoforms migrate as a doublet of ~50-55 kDa upon Western blot analysis.

  

562590 Rev. 1
Format Details
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PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
altImg
PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
562590 Rev.1
Citations & References
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Development References (7)

  1. Carter RS, Pennington KN, Ungurait BJ, Ballard DW. In vivo identification of inducible phosphoacceptors in the IKKgamma/NEMO subunit of human IkappaB kinase. J Biol Chem. 2003; 278(22):19642-19648. (Biology). View Reference
  2. DiDonato JA, Hayakawa M, Rothwarf DM, Zandi E, Karin M. A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB. Nature. 1997; 388(6642):548-554. (Biology). View Reference
  3. Ling L, Cao Z, Goeddel DV. NF-kappaB-inducing kinase activates IKK-alpha by phosphorylation of Ser-176. Proc Natl Acad Sci U S A. 1998; 95(7):3792-3797. (Biology). View Reference
  4. Mercurio F, Zhu H, Murray BW, et al. IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation. Science. 1997; 278(5339):860-866. (Biology). View Reference
  5. Nakano H, Shindo M, Sakon S, et al. Differential regulation of IkappaB kinase alpha and beta by two upstream kinases, NF-kappaB-inducing kinase and mitogen-activated protein kinase/ERK kinase kinase-1. Proc Natl Acad Sci U S A. 1998; 95(7):3537-3542. (Biology). View Reference
  6. Rothwarf DM, Zandi E, Natoli G, Karin M. IKK-gamma is an essential regulatory subunit of the IkappaB kinase complex. Nature. 1998; 395(6699):297-300. (Biology). View Reference
  7. Shifera AS. The zinc finger domain of IKKγ (NEMO) protein in health and disease. J Cell Mol Med. 2010; 14(10):2404-2414. (Biology). View Reference
View All (7) View Less
562590 Rev. 1

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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

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