Human/Mouse Phospho-STAT3 (Y705) 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 and mouse STAT3 (Y705) in cell lysates. An immobilized capture antibody specific for STAT3 binds both phosphorylated and unphosphorylated STAT3. 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: STAT3

The STAT family of transcription factors, first described as key mediators of interferon signal transduction, are now recognized as signaling factors downstream of many cytokines, growth factors, and hormones. At least seven STATs exist: STAT1, 2, 3, 4, 5a, 5b, and 6. They are responsible for an array of cellular activities including regulating growth, survival, differentiation, motility, and the immune response. They are characterized structurally by the presence of the Src homology 2 (SH2) domain, and a C-terminal tyrosine phosphorylation site. Upon ligand binding, they are recruited to the activated receptor, via their SH2 domains, to receptor-associated tyrosine kinases of the Janus kinase family (JAK). Once phosphorylated by JAK, STATS are free to hetero- or homo-dimerize, and translocate to the nucleus where they bind DNA to regulate gene transcription.

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.