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Alexa Fluor® 647 Mouse anti-TBK1 (pS172)
Alexa Fluor® 647 Mouse anti-TBK1 (pS172)

Analysis of TBK1 (pS172) in transfected human epithelial cells.  The 293 fetal kidney cell line was either co-transfected with TBK1 and IRF-7 expression vectors (dashed histogram) or un-transfected (solid line).  After 24 hours, the cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with Alexa Fluor® 647 Mouse anti-TBK1 (pS172, Cat. No. 558603).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Analysis of TBK1 (pS172) in transfected human epithelial cells.  The 293 fetal kidney cell line was either co-transfected with TBK1 and IRF-7 expression vectors (dashed histogram) or un-transfected (solid line).  After 24 hours, the cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with Alexa Fluor® 647 Mouse anti-TBK1 (pS172, Cat. No. 558603).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Product Details
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BD Phosflow™
T2K, NAK
Human (QC Testing), Mouse (Predicted)
Mouse IgG1, κ
Phosphorylated Human TBK1
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_647128
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 to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was 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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  3. 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.
  4. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  5. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  6. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
  7. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
558603 Rev. 2
Antibody Details
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J133-587

NF-κB is a ubiquitously expressed transcription factor that regulates many cytokine and Ig genes.  It is involved in immune, inflammatory, viral, and acute phase responses.  In most cells, NF-κB is sequestered in an inactive cytoplasmic form via interactions with the inhibitory proteins IκBα, IκBβ, and IκBε.  Stimulation induces the release, activation, and nuclear translocation of NF-κB.  Release of NF-κB results from the phosphorylation and proteolytic degradation of the IκB proteins.  Two cytokine-inducible IκB kinases (IKKα and IKKβ) phosphorylate and target the IκB proteins for degradation via the ubiquitin pathway.  IKKγ/NEMO, a third member of the IKK complex, functions as a regulatory subunit and interacts directly with IKKβ.  TBK1 (TANK-binding kinase 1, also known as T2K or NAK), a protein of 729 amino acids, is another member of the IKK family of kinases regulating NF-κB downstream of the tumor necrosis factor and Toll-like receptor pathways.  TBK1 forms a complex with the adaptor proteins TANK (TRAF-associated NF-κB activator) and TRAF2 (TNF-receptor-associated factor 2), and this oligomer is required for activation and phosphorylation of TBK1 at serine 172 (S172).

The J133-587 monoclonal antibody recognizes the phosphorylated S172 of human TBK1.  Our in-house testing is performed on a cell line that has been co-transfected with TBK1 and IRF-7 because we have been unable to detect endogenous TBK1 (pS172) by western blotting of lysates from activated cell lines (HeLa activated with PMA and A431 activated with PDGF).  In the transfectants, the over-expression of TBK1 is necessary for phosphorylation of IRF-7, but the presence of IRF-7 does not affect TBK1 expression or phosphorylation.  We confirmed that mAb J133-587 does not cross-react with IRF-7 by ELISA.

558603 Rev. 2
Format Details
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Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
558603 Rev.2
Citations & References
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Development References (2)

  1. Kishore N, Huynh QK, Mathialagan S, et al. IKK-i and TBK-1 are enzymatically distinct from the homologous enzyme IKK-2: comparative analysis of recombinant human IKK-i, TBK-1, and IKK-2.. J Biol Chem. 2002; 277(16):13840-13847. (Biology). View Reference
  2. Viatour P, Merville M-P, Bours V, Chariot A. Phosphorylation of NF-kappaB and IkappaB proteins: implications in cancer and inflammation. Trends Biochem Sci. 2005; 30(1):43-52. (Biology). View Reference
558603 Rev. 2

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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.