Description
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.
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.
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.
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.
