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Nur77 (D63C5) XP® Rabbit mAb (PE Conjugate) #59999
Gallery: Nur77 (D63C5) XP® Rabbit mAb (PE Conjugate) #59999
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
Nur77 (D63C5) XP® Rabbit mAb (PE Conjugate) detects endogenous levels of total human Nur77 protein.Species Reactivity: Human
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu535 of human Nur77.
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 Nur77 (D63C5) XP® Rabbit mAb #3960.
Nur77, also known as TR3 and NGFI-B, is an immediate-early response gene and an orphan member of the steroid/thyroid/retinoid receptor superfamily (1-3). Nur77 is composed of an amino-terminal transactivation domain, a central DNA-binding domain and a carboxy-terminal ligand-binding domain. Expression of Nur77 is rapidly induced by a variety of stimuli, including apoptotic, mitogenic and stress signals (1-6). It has been proposed to have many functions related to cell proliferation, differentiation and apoptosis. Nur77 has been extensively studied in T cells where it has been implicated in the process of negative selection and TCR-mediated apoptosis (5,6). Nur77 binds to specific DNA elements leading to the regulation of target genes (7). As a possible mechanism for regulating apoptosis, Nur77 can induce the expression of apoptotic genes such as FasL and TRAIL (8,9). Nur77 is heavily phosphorylated by multiple kinases, which may affect its transactivation activity as well as its subcellular localization (4,10,11). Translocation of Nur77 from the nucleus to the mitochondria can regulate its association with Bcl-2 and control the release of cytochrome c, thereby triggering apoptosis (12,13).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.