Phospho-SHP-2 (Y542) DuoSet IC ELISA

Name: Phospho-SHP-2 (Y542) DuoSet IC ELISA
Brand: R&D Systems
SKU: DYC3790-2

R&D Systems | Catalog # DYC3790-2

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Product Documents

Discontinued Product

DYC3790-2 has been discontinued. View all SHP-2 products.

Product Summary
Product Specifications
Scientific Data
Kit Contents
Other Reagents Required
Preparation & Storage
Background
Product Documents
Specific Notices
Research Areas

Key Product Details

Assay Type

Solid Phase Sandwich ELISA

Assay Range

156-10000 pg/mL

Sample Type

Cell Lysates

Reactivity

Human, Mouse, Rat

Phospho-SHP-2 (Y542) DuoSet IC ELISA Features

Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
Development protocols are provided to guide further assay optimization
Assay can be customized to your specific needs
Available in 2, 5, and 15- (96-well) plate pack sizes
Economical alternative to Western blot

View all SHP-2 ELISA Kits »

Product Summary for Phospho-SHP-2 (Y542) DuoSet IC ELISA

This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure phosphorylated human, mouse, and rat SHP-2 (Y542) in cell lysates. An immobilized capture antibody specific for SHP-2 binds both phosphorylated and unphosphorylated SHP-2. After washing away unbound material, a biotinylated detection antibody is used to detect only phosphorylated receptor, utilizing a standard HRP format.

Product Specifications

Assay Format

96-well strip plate (sold separately)

Sample Volume Required

Cell lysates (100 µL)

Detection Method

Colorimetric ELISA - 450nm (TMB)

Conjugate

Biotin

Specificity

Please see the product datasheet

Label

HRP

Scientific Data Images for Phospho-SHP-2 (Y542) DuoSet IC ELISA

Standard Curve

The Human/Mouse/Rat Phospho-SHP-2 (Y542) DuoSet IC ELISA specifically recognizes SHP-2 phosphorylated at Y542 as shown by Western blot analysis of the protein immunoprecipitated with the capture antibody supplied in the kit.

A lysate from Jurkat human acute T cell leukemia cells was immunoprecipitated with the Phospho-SHP-2 (Y542) Capture Antibody. Unbound material was removed by washing. The bound material was solubilized in SDS-PAGE loading buffer and subjected to Western blotting using either anti-SHP-1 (Catalog # AF1878) or anti-SHP-2 (Catalog # AF1894) polyclonal antibodies. Both SHP-1 and SHP-2 were found in the original lysate but only SHP-2 was immunoprecipitated. SHP-1, which is 54% homologous to SHP-2, remained in the post-immunoprecipitation (IP) lysate.To further determine specificity, unphosphorylated recombinant human SHP-1 and SHP-2 were assayed at 100 ng/mL and did not cross-react or interfere in the assay.

Growth Factor Induction of Phospho-SHP-2 (Y542) in Mouse Cells.

NIH-3T3 mouse embryonic fibroblast cells were treated with 50 ng/mL of recombinant human PDGF-BB (Catalog # 220-BB) for 20 minutes. The lysates were either quantified with this DuoSet® IC ELISA or immunoblotted with the rabbit anti-SHP-2 antibody provided in this kit or total SHP-2 antibody (Catalog # AF1894). The PDGF-BB treatment caused a greater than five-fold induction of hospho-SHP-2 (Y542) as measured using the ELISA. In the Western Blots, a marked induction of phospho-SHP-2 (Y542) was detected without a change in the levels of total SHP-2 protein.

Kit Contents for Phospho-SHP-2 (Y542) DuoSet IC ELISA

Capture Antibody
Conjugated Detection Antibody
Calibrated Immunoassay Standard or Control
Streptavidin-HRP

Other Reagents Required

PBS:

(Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂O₄, pH 7.2 - 7.4, 0.2 µm filtered

Wash Buffer: (Catalog # WA126), or equivalent

Lysis Buffer*

IC Diluent*

Blocking Buffer*

Substrate Solution: 1:1 mixture of Color Reagent A (H₂O₂) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)

Stop Solution: 2 N H₂SO₄ (Catalog # DY994)

Microplates: From Costar EIA Plate (Costar Catalog # 2592) or R&D Systems (Catalog # DY990), or equivalent

Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent

*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product

Preparation and Storage

Shipping

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: SHP-2

The SH2 domain-containing phosphatases, SHP-1 and SHP-2, are well-studied cytosolic tyrosine phosphatases that share many structural and regulatory features. They both have two tandem SH2 domains at the N-terminus, followed by a catalytic domain, and an inhibitory C-terminus. Despite their structural similarity, the enzymes appear to have different physiological roles and exhibit different expression patterns. SHP-1 is highly expressed in hematopoietic tissues, and in general has a negative impact on lymphocyte signaling. In contrast, SHP-2 is widely expressed, and in general promotes signaling pathways that lead to differentiation, cell growth, or migration.