Description
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.
Format
- Format
Alexa Fluor® 647
- Excitation Source
Red 633 nm
- Excitation Max
650 nm
- Emission Max
668 nm
Alexa Fluor® 647 conjugates are highly photostable and remain fluorescent over a broad pH range. The excitation and emission maxima are nearly identical to those of APC. However, APC tends to be brighter while Alexa Fluor® 647 is more optimal for intracellular applications. This fluorochrome exhibits uncommon photostability, making it an ideal choice for use in fluorescence microscopy. Due to nearly identical excitation and emission properties but different spillover characteristics, APC and Alexa Fluor® 647 cannot be used simultaneously.
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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
- 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).
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
