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BD Phosflow™ Alexa Fluor® 488 Mouse anti-TBK1
Clone 637Ig11.2 (RUO)
Analysis of TBK1 in transfected human epithelial cells. The 293 fetal kidney cell line (left panel) 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® 488 Mouse anti-TBK1 (Cat. No. 558697). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system. The specificity of mAb 637Ig11.2 was confirmed by western blot (right panel) using unconjugated antibody on lysates from untransfected (left panel) and TBK1/IRF-7 co-transfected (right panel) 293 cells. The TBK1-Myc tagged fusion protein is identified as a band of 84 kDa in the lysate of co-transfected cells.
Analysis of TBK1 in transfected human epithelial cells. The 293 fetal kidney cell line (left panel) 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® 488 Mouse anti-TBK1 (Cat. No. 558697). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system. The specificity of mAb 637Ig11.2 was confirmed by western blot (right panel) using unconjugated antibody on lysates from untransfected (left panel) and TBK1/IRF-7 co-transfected (right panel) 293 cells. The TBK1-Myc tagged fusion protein is identified as a band of 84 kDa in the lysate of co-transfected cells.
Analysis of TBK1 in transfected human epithelial cells. The 293 fetal kidney cell line (left panel) 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® 488 Mouse anti-TBK1 (Cat. No. 558697). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system. The specificity of mAb 637Ig11.2 was confirmed by western blot (right panel) using unconjugated antibody on lysates from untransfected (left panel) and TBK1/IRF-7 co-transfected (right panel) 293 cells. The TBK1-Myc tagged fusion protein is identified as a band of 84 kDa in the lysate of co-transfected cells.
Analysis of TBK1 in transfected human epithelial cells. The 293 fetal kidney cell line (left panel) 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® 488 Mouse anti-TBK1 (Cat. No. 558697). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system. The specificity of mAb 637Ig11.2 was confirmed by western blot (right panel) using unconjugated antibody on lysates from untransfected (left panel) and TBK1/IRF-7 co-transfected (right panel) 293 cells. The TBK1-Myc tagged fusion protein is identified as a band of 84 kDa in the lysate of co-transfected cells.
Analysis of TBK1 in transfected human epithelial cells. The 293 fetal kidney cell line (left panel) 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® 488 Mouse anti-TBK1 (Cat. No. 558697). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system. The specificity of mAb 637Ig11.2 was confirmed by western blot (right panel) using unconjugated antibody on lysates from untransfected (left panel) and TBK1/IRF-7 co-transfected (right panel) 293 cells. The TBK1-Myc tagged fusion protein is identified as a band of 84 kDa in the lysate of co-transfected cells.
Analysis of TBK1 in transfected human epithelial cells. The 293 fetal kidney cell line (left panel) 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® 488 Mouse anti-TBK1 (Cat. No. 558697). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system. The specificity of mAb 637Ig11.2 was confirmed by western blot (right panel) using unconjugated antibody on lysates from untransfected (left panel) and TBK1/IRF-7 co-transfected (right panel) 293 cells. The TBK1-Myc tagged fusion protein is identified as a band of 84 kDa in the lysate of co-transfected cells.
Product Details
BD Phosflow™
T2K, NAK
Human (QC Testing)
Mouse IgG1, κ
Human TBK1 C terminus Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645377
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® 488 under optimum conditions, and unreacted Alexa Fluor® 488 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).
- Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Fixation Buffer RUO
Size
100 ML
Cat No.
554655
Perm Buffer III RUO
Size
125 ML
Cat No.
558050
Alexa Fluor® 488 Mouse IgG1 κ Isotype Control RUO
Size
50 Tests
Cat No.
557782
558697 Rev. 3
Antibody Details
637Ig11.2
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.
The 637Ig11.2 monoclonal antibody recognizes human TBK1, regardless of phosphorylation status. 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 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 637Ig11.2 does not cross-react with IRF-7 by western blot analysis using the purified antibody.
558697 Rev. 3
Format Details
Alexa Fluor™ 488
Alexa Fluor™ 488 Dye is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 517-nm. Alexa Fluor™ 488 is designed to be excited by the Blue laser (488 nm) and detected using an optical filter centered near 520-nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 488
494 nm
517 nm
558697 Rev.3
Citations & References
View product citations for antibody "558697" on CiteAb
Development References (2)
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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
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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
558697 Rev. 3
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
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.