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).
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.
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.
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised August 2019
Protocol Id: 407
Phospho-SHP-2 (Tyr580) (D66F10) Rabbit mAb detects endogenous level of SHP-2 only when phosphorylated at Tyr580.Species Reactivity:
Mouse, RatSpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr580 of human SHP-2 protein.
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).
Explore pathways + proteins related to this product.
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