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Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb (PE Conjugate) #11979
Flow cytometric analysis of HT-1080 cells, untreated (blue) or treated with hTGF-β3 #8425 (10 ng/ml, 30 min; green), using Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb (PE Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).Learn more about how we get our images
Gallery: Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb (PE Conjugate) #11979
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-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb (PE Conjugate) recognizes endogenous levels of Smad2 protein when phosphorylated at Ser465 and Ser467. This antibody also recognizes endogenous levels of Smad3 protein when phosphorylated at Ser422 only or at both Ser423 and Ser425.Species Reactivity: Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser465/467 of human Smad2 protein.
This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb #8828.
Members of the Smad family of signal transduction molecules are components of a critical intracellular pathway that transmit TGF-β signals from the cell surface into the nucleus. Three distinct classes of Smads have been defined: the receptor-regulated Smads (R-Smads), which include Smad1, 2, 3, 5, and 8; the common-mediator Smad (co-Smad), Smad4; and the antagonistic or inhibitory Smads (I-Smads), Smad6 and 7 (1-5). Activated type I receptors associate with specific R-Smads and phosphorylate them on a conserved carboxy terminal SSXS motif. The phosphorylated R-Smad dissociates from the receptor and forms a heteromeric complex with the co-Smad (Smad4), allowing translocation of the complex to the nucleus. Once in the nucleus, Smads can target a variety of DNA binding proteins to regulate transcriptional responses (6-8).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.