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13328S 100 µl (50 tests) $299.00.0
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REACTIVITY SENSITIVITY MW (kDa) Isotype
M R Endogenous Rabbit IgG
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Flow Cytometry

Flow cytometric analysis of NIH/3T3 cells, untreated (blue) or treated with Human Platelet-Derived Growth Factor BB (hPDGF-BB) #8912 (green), using Phospho-SHP-2 (Tyr580) (D66F10) Rabbit mAb (PE Conjugate).

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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

Phospho-SHP-2 (Tyr580) (D66F10) Rabbit mAb detects endogenous level of SHP-2 only when phosphorylated at Tyr580.


Species Reactivity: Mouse, Rat
Species predicted to react based on 100% sequence homology: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr580 of human SHP-2 protein.

Product Description

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in mouse cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-SHP-2 (Tyr580) (D66F10) Rabbit mAb #5431.


SHP-2 (PTPN11) is a ubiquitously expressed, nonreceptor protein tyrosine phosphatase (PTP). It participates in signaling events downstream of receptors for growth factors, cytokines, hormones, antigens, and extracellular matrices in the control of cell growth, differentiation, migration, and death (1). Activation of SHP-2 and its association with Gab1 is critical for sustained Erk activation downstream of several growth factor receptors and cytokines (2). In addition to its role in Gab1-mediated Erk activation, SHP-2 attenuates EGF-dependent PI3 kinase activation by dephosphorylating Gab1 at p85 binding sites (3). SHP-2 becomes phosphorylated at Tyr542 and Tyr580 in its carboxy-terminus in response to growth factor receptor activation (4). These phosphorylation events are thought to relieve basal inhibition and stimulate SHP-2 tyrosine phosphatase activity (5). Mutations in the corresponding gene result in a pair of clinically similar disorders (Noonan syndrome and LEOPARD syndrome) that may result from abnormal MAPK regulation (6).


1.  Qu, C.K. (2000) Cell Res 10, 279-88.

2.  Maroun, C.R. et al. (2000) Mol Cell Biol 20, 8513-25.

3.  Zhang, S.Q. et al. (2002) Mol Cell Biol 22, 4062-72.

4.  Bennett, A.M. et al. (1994) Proc Natl Acad Sci USA 91, 7335-9.

5.  Lu, W. et al. (2001) Mol Cell 8, 759-69.

6.  Edouard, T. et al. (2007) Cell Mol Life Sci 64, 1585-90.


Entrez-Gene Id 5781
Swiss-Prot Acc. Q06124


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

13328
Phospho-SHP-2 (Tyr580) (D66F10) Rabbit mAb (PE Conjugate)