Revvity, 64ALKTPEH, HTRF Total ALK 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
Total ALK assay principle
The Total-ALK assay quantifies the expression level of ALK in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-ALK assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of ALK 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 ALK 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 addition of the Total-ALK HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total ALK one-plate assay protocol
Detection of Total ALK 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 1 Lysis Buffer 2 Lysis Buffer 4
Molecular Modification-Total
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