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Alexa Fluor® 647 Mouse anti-Human IRAK4
Alexa Fluor® 647 Mouse anti-Human IRAK4

Analysis of IRAK4 in activated human peripheral blood lymphocytes (far left panel). Human peripheral blood mononuclear cells (PBMC) were lysed and fixed with 1X BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10 minutes at 37ºC, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, and then stained with either Alexa Fluor® 647 Mouse IgG1 κ Isotype control (Cat. No. 557783, shaded histogram) or Alexa Fluor® 647 Mouse anti-Human IRAK4 (Cat. No. 560315, open histogram).  For data analysis, lymphocytes were selected by scatter profile.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Analysis of IRAK4 in human epithelioid carcinoma (center left panel).  HeLa S3 cells (ATCC CCL 2.2) were either transfected with IRAK4 RNAi-transfected HeLaS3 cells (open histogram) or untreated (shaded histogram).  The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III) on ice for 30 minutes, and then stained with Alexa Fluor® 647 Mouse anti-Human IRAK4. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Confirmation of the specificity of mAb L29-525 by western blot analysis (center right panel).  Lysates of human PBMC were probed with unconjugated antibody at a concentration of 0.1 µg/ml.  IRAK4 is identified as a band of about 52 kDa.

Western blot validation of IRAK4 by RNAi (far right panel).  Lysates from HeLa S3 cells (lane 1) and IRAK4 RNAi-transfected HeLa S3 cells (lane 2) were probed with unconjugated mAb L29-525 at 0.015 µg/ml.  Down-regulation of IRAK4 expression is evident in the RNAi-transfected cells.  

Analysis of IRAK4 in activated human peripheral blood lymphocytes (far left panel). Human peripheral blood mononuclear cells (PBMC) were lysed and fixed with 1X BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10 minutes at 37ºC, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, and then stained with either Alexa Fluor® 647 Mouse IgG1 κ Isotype control (Cat. No. 557783, shaded histogram) or Alexa Fluor® 647 Mouse anti-Human IRAK4 (Cat. No. 560315, open histogram).  For data analysis, lymphocytes were selected by scatter profile.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Analysis of IRAK4 in human epithelioid carcinoma (center left panel).  HeLa S3 cells (ATCC CCL 2.2) were either transfected with IRAK4 RNAi-transfected HeLaS3 cells (open histogram) or untreated (shaded histogram).  The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III) on ice for 30 minutes, and then stained with Alexa Fluor® 647 Mouse anti-Human IRAK4. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Confirmation of the specificity of mAb L29-525 by western blot analysis (center right panel).  Lysates of human PBMC were probed with unconjugated antibody at a concentration of 0.1 µg/ml.  IRAK4 is identified as a band of about 52 kDa.

Western blot validation of IRAK4 by RNAi (far right panel).  Lysates from HeLa S3 cells (lane 1) and IRAK4 RNAi-transfected HeLa S3 cells (lane 2) were probed with unconjugated mAb L29-525 at 0.015 µg/ml.  Down-regulation of IRAK4 expression is evident in the RNAi-transfected cells.  

Product Details
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BD Phosflow™
IRAK-4, REN64, Q69FE3
Human (QC Testing)
Mouse BALB/c IgG1, κ
Human IRAK4 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645422
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.

Recommended Assay Procedures

This antibody conjugate is suitable for intracellular staining of human peripheral blood mononuclear cells and cell lines (using BD Phosflow™ Lyse/Fix Buffer or BD Cytofix™ Fixation Buffer), but not  human whole blood.  BD Phosflow™ Perm Buffer II or Perm Buffer III may be used.

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. An isotype control should be used at the same concentration as the antibody of interest.
  3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  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. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. 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.
  8. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  9. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560315 Rev. 2
Antibody Details
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L29-525

Interleukin 1 (IL-1) is a proinflammatory cytokine that mediates the host inflammatory response. IL-1 exerts its biological effects through its type I receptor (IL-1RI), which is found on most cell types. When IL-1 binds to the IL-1RI, NF-κB (transcription factor) is activated and translocates to the nucleus where it induces gene expression. IL-1-receptor-associated kinase (IRAK) has been reported to co-immunoprecipitate with IL-1RI. The IRAK family is made up of four identified Pelle homologs that regulate the activation of NF-κB and mitogen-activated protein (MAP) kinase pathways. IRAK4 is a unique member of the IRAK family because it is the closest mammalian homolog to Pelle. Also, when overexpressed, its kinase activity allows it to activate both the NF-κB and mitogen-activated protein (MAP) kinase pathways, and block the IL-1-induced activation and modification of IRAK1. IRAK4 is also able to phosphorylate IRAK1, which suggests that IRAK4 affects the early signal transduction of Toll/IL-1 receptors before the involvement of IRAK1.

The L29-525 monoclonal antibody recognizes human IRAK4, regardless of phosphorylation status.  The specificity of this antibody conjugate for flow cytometric analysis was validated by confirming that RNA-mediated interference (RNAi) of the specific protein reduced the staining of the cells (see figure).  Furthermore, the capacity of the RNAi to down-regulate the expression of the relevant protein was confirmed by western blot analysis.

560315 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
560315 Rev.2
Citations & References
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Development References (4)

  1. Hatao F, Muroi M, Hiki N, et al. Prolonged Toll-like receptor stimulation leads to down-regulation of IRAK-4 protein. J Leukoc Biol. 2004; 76(4):904-908. (Biology). View Reference
  2. Li S, Strelow A, Fontana EJ, Wesche H. IRAK-4: a novel member of the IRAK family with the properties of an IRAK-kinase. Proc Natl Acad Sci U S A. 2002; 99(8):5567-5572. (Biology). View Reference
  3. Lye E, Mirtsos C, Suzuki N, Suzuki S, Yeh WC. The role of interleukin 1 receptor-associated kinase-4 (IRAK-4) kinase activity in IRAK-4-mediated signaling. J Biol Chem. 2004; 279(39):40653-40658. (Biology). View Reference
  4. Suzuki N, Saito T. IRAK-4 – a shared NF-κB activator in innate and acquired immunity. Trends Immunol. 2006; 27(12):566-572. (Biology). View Reference
View All (4) View Less
560315 Rev. 2

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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.