Upstream / Downstream

pathwayImage

Explore pathways related to this product.

To Purchase # 52478S

52478S 100 µl (50 tests) $299.00.0
$0.00

Questions?

Find answers on our FAQs page.

ANSWERS  

PhosphoSitePlus® Resource

  • Additional protein information
  • Analytical tools

LEARN MORE

REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
Image
Image
Image
Image

Flow Cytometry

Flow cytometric analysis of K-562 cells (blue) and THP-1 cells (green) using IRF-5 (E1N9G) Rabbit mAb (PE Conjugate).

Learn more about how we get our images
Image
Image
Page

Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. 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

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Product Usage Information

Application Dilutions
Flow Cytometry 1:50

Storage: Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibodies. Protect from light. Do not freeze.

Specificity / Sensitivity

IRF-5 (E1N9G) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total IRF-5 protein.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu169 of human IRF-5 protein.

Product Description

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 IRF-5 (E1N9G) Rabbit mAb #13496.


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-5 is expressed in lymphoid tissues and peripheral blood lymphocytes and participates in the induction of type I interferon genes following viral infection (3). Activation of IRF-5 signaling is triggered by components of the toll-like receptor (TLR) pathway, including TLR7 and MyD88 (4,5). Research studies show that genetic variants of IRF5 have been associated with disorders where the IFN pathway is abnormally activated, such as systemic lupus erythematosus (6,7).


1.  Taniguchi, T. et al. (2001) Annu Rev Immunol 19, 623-55.

2.  Honda, K. and Taniguchi, T. (2006) Nat Rev Immunol 6, 644-58.

3.  Barnes, B.J. et al. (2001) J Biol Chem 276, 23382-90.

4.  Takaoka, A. et al. (2005) Nature 434, 243-9.

5.  Schoenemeyer, A. et al. (2005) J Biol Chem 280, 17005-12.

6.  Sigurdsson, S. et al. (2005) Am J Hum Genet 76, 528-37.

7.  Graham, R.R. et al. (2006) Nat Genet 38, 550-5.


Entrez-Gene Id 3663
Swiss-Prot Acc. Q13568


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

52478
IRF-5 (E1N9G) Rabbit mAb (PE Conjugate)