Revvity, 64ALKY04PEH, HTRF Phospho-ALK (Tyr1604) Detection Kit, 10,000 Assay Points

Overview
ALK (anaplastic lymphoma kinase) is a transmembrane tyrosine kinase belonging to the superfamily of insulin receptors. ALK regulates several pathways involved in cell survival, proliferation, and cell cycling, including the AKT/PI3K,MAPK/ERK, STAT3 and NFkB pathways.The ALK ligands, pleiotrophin (PTN) and midkine(MDK), induce ALK phosphorylation and activation on  multiple Tyrosine including Y1604. ALK deregulation is associated with  various cancers such as NSCLC, and neuroblastomas. Indeed, oncogenic ALK is driven by i) gene translocations and subsequent intracellular ALK fusion proteins (eg, EML4-ALK, NPM1-ALK, TPM3-ALK, VCL-ALK); ii) mutations (eg G263, R401, R551, P968 and E1242). Oncogenic ALK is constitutive active which results in uncontroled activation of downstream signaling pathways, leading to tumor cells survival and proliferation.
How it works
Phospho-ALK (Tyr1604) assay principle
The HTRF Phospho-ALK (Tyr1604) assay measures ALK when phosphorylated at Tyr1604. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-ALK (Tyr1604) assay uses 2 labeled antibodies: one with a donor fluorophore, the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving the two 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-ALK (Tyr1604) two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the additon of the Phospho-ALK Tyr1604) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-ALK (Tyr1604) one-plate assay protocol
Detection of  Phospho-ALK (Tyr1604) 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 4
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Technology-TR-FRET
Unit Size-10,000 assay points