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Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb (PE Conjugate) #83611
Gallery: Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb (PE Conjugate) #83611
Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- 16% Formaldehyde (methanol free).
- 100% methanol.
- Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
- Fix for 15 min at room temperature.
- Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.
- Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
- Incubate 30 min on ice.
- Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.
- Aliquot desired number of cells into tubes or wells.
- Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
- Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
- Incubate for 1 hr at room temperature. Protect from light.
- Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
- Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).
E. Optional DNA Dye
- Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
- Incubate for at least 5 min at room temperature.
- Analyze cells in DNA staining solution on flow cytometer.
posted July 2009
revised June 2017
Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb (PE Conjugate) recognizes endogenous levels of IRF-3 protein only when phosphorylated at Ser396.Species Reactivity: Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser396 of human IRF-3 protein.
This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb #29047.
Interferon regulatory factors (IRFs) comprise a family of transcription factors that function within the Jak/Stat pathway to regulate interferon (IFN) and IFN-inducible gene expression in response to viral infection (1). IRFs play an important role in pathogen defense, autoimmunity, lymphocyte development, cell growth, and susceptibility to transformation. The IRF family includes nine members: IRF-1, IRF-2, IRF-9/ISGF3γ, IRF-3, IRF-4 (Pip/LSIRF/ICSAT), IRF-5, IRF-6, IRF-7, and IRF-8/ICSBP. All IRF proteins share homology in their amino-terminal DNA-binding domains. IRF family members regulate transcription through interactions with proteins that share similar DNA-binding motifs, such as IFN-stimulated response elements (ISRE), IFN consensus sequences (ICS), and IFN regulatory elements (IRF-E) (2).
IRF-3 can inhibit cell growth and plays a critical role in controlling the expression of genes in the innate immune response (1-4). In unstimulated cells, IRF-3 is present in the cytoplasm. Viral infection results in phosphorylation of IRF-3 and leads to its translocation to the nucleus where it activates promoters containing IRF-3-binding sites. Phosphorylation of IRF-3 occurs at a cluster of C-terminal serine and threonine residues (between 385 and 405) leading to its association with the p300/CBP coactivator protein that promotes DNA binding and transcriptional activity (5). During infection, IRF-3 is likely activated through a pathway that includes activation of Toll-like receptors and of a kinase complex that includes IKKε and TBK1 (6,7). IRF-3 is phosphorylated at Ser396 following viral infection, expression of viral nucleocapsid, and double stranded RNA treatment. These events likely play a role in the activation of IRF-3 (8).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.