Human/Mouse/Rat Phospho-SHP-2 (Y542) DuoSet IC ELISA Summary

Product Summary

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.

Preparation and Storage

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.

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.     Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.     Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.     Add 50 μL of Assay Diluent to each well.

4.     Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.     Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.     Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.     Aspirate and wash 5 times.

8.     Add 100 μL Substrate Solution to each well.

9.     Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.