Revvity, 64SYKY525PEG, HTRF Human Phospho-SYK (Tyr525/526) Detection Kit, 500 Assay Points

Overview
This HTRF cell-based assay conveniently and accurately detects phosphorylated SYK at Tyr525/526. ​​​​​​​
SYK (Spleen Tyrosine Kinase) is a cytoplasmic tyrosine/serine kinase which plays a crucial role in signal transduction, especially in immune cells such as B cells, macrophages, microglia (brain-resident macrophages), neutrophils and mast cells. Upon cell stimulation, SYK interacts with the cytoplasmic domains of activated immune receptors and binds to the specific phosphorylated receptor motifs (called "p-ITAMs"), leading to its activation by auto-phosphorylation of its catalytic domain at Tyr525/526.
SYK phosphorylation is a readout of key inflammatory pathways such as BCR, FcR and TREM2 signaling pathways that are altered in various pathologies such as auto-immune diseases, neurodegeneration, cancers and allergic disorders.
How it works
Phospho-SYK (Tyr525/526) assay principle
The Phospho-SYK (Tyr525/526) assay measures SYK when phosphorylated at Tyr525/526. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies, and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Phospho-SYK (Tyr525/526) two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Phospho-SYK (Tyr525/526) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-SYK (Tyr525/526) one-plate assay protocol
Detection of Phosphorylated SYK (Tyr525/526) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 3 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Therapeutic Area-Neuroscience Oncology & Inflammation
Unit Size-500 assay points