Revvity, 64SYKTPEH, HTRF Human Total SYK Detection Kit, 10,000 Assay Points

Overview
The Total SYK cellular assay monitors total SYK, and can be used as a normalization assay with our phospho-SYK (Tyr525/526) kit. This kit is compatible with the buffers from the phospho-SYK kit, so the same lysate can be used for analyses of both the phosphorylated and the total protein populations.
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
Total-SYK assay principle
The Total SYK assay quantifies the expression level of SYK in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-SYK assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of SYK in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein's expression under a no-wash assay format.

Total-SYK 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 Total-SYK HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-SYK one-plate assay protocol
Detection of Total SYK 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 2 Lysis Buffer 3
Molecular Modification-Total
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-10,000 assay points