HTRF Phospho-ALK (Tyr1604) Detection Kit, 500 Assay Points

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.

Comparison between HTRF and WB sensitivity on Phospho Y1604 ALK

SU-DHL-1 cells were cultured in a T175 flask in complete medium at 37°C, 5% CO2.

After centrifugation and medium removal, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1x) for 30 min at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer #4 (1x), and 16µL of pure sample. Each dilution was transferred into a 384-well small volume microplate before the addition of 4µL of HTRF Phospho Y1604 ALK detection antibodies. The HTRF signals were recorded after an overnight incubation.

Equal amounts of lysates were loaded into a gel for a side by side comparison between HTRF and Western Blot.

In these conditions, the HTRF Phospho Y1604 ALK assay is 64-fold more sensitive than the Western Blot.

ALK (anaplastic lymphoma kinase) is a transmembrane tyrosine kinase which regulates cell survival, proliferation, and cell cycling, The ALK ligands, pleiotrophin (PTN) and midkine (MDK), induce ALK phosphorylation and activation on multiple Tyrosine including Y1604 which activates multiple signaling pathways such as AKT/PI3K,MAPK/ERK, STAT3 and NFkB pathways.

Constitutive activation of oncogenic ALK fusion proteins or mutated ALK is associated with various cancers such as NSCLC, and neuroblastomas.