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.

Specificity of HTRF Total ALK assay using siRNA

IMR-32 cells were plated in a 96-well culture plates at a density of 50,000 cells/well and incubated for 24 hours at 37°C, 5% CO2. The cells were then transfected with ON-TARGETplus SMARTPool siRNAs specific for ALK, as well as with a non-targeting siRNA as a negative control. After a 24h incubation with siRNAs, the medium was renewed for an additional 24h incubation and the cells were lysed with 50 µL of supplemented lysis buffer #4 for 30min.

Then 16 µL of lysates were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Total ALK detection antibodies. An additional 4 µL of lysates (supplemented with 12 µL diluent #8) were also transferred into the microplate to monitor the alpha-tubulin level using the HTRF Alpha-tubulin Housekeeping Cellular Kit (64ATUBPET/G/H). The HTRF signal was recorded after an overnight incubation.

siRNA targeting ALK led to a 53% signal decrease compared to control cells transfected with the negative siRNA.  The alpha tubulin level remained stable.

Total ALK assay versatility on human cell lines expressing wild type or mutated ALK

The experiments were carried out using the two-plate assay protocol for adherent cells. The human cell lines SK-N-SH expressing the F1174L mutation of the ALK protein and IMR32 expressing ALK wild type were seeded in a 96-well culture plate (100,000 cells/well in complete culture medium) and cultured at 37°C, 5% CO2 for 24 hours.

The day after, the cells were lysed with 50 µL of supplemented lysis buffer #4, and 16 µL of lysate were transferred into a ProxiPlate-384 Plus, 6008280/9) followed by the addition of 4 µL of HTRF Total ALK kit detection antibodies. In parallel, 4 µL of lysates (supplemented with 12 µL diluent #8) were transferred into the microplate, and alpha-tubulin housekeeping protein was detected with the HTRF Alpha-tubulin Housekeeping Cellular Kit (64ATUBPET/G/H). The HTRF signal was recorded after an overnight incubation.

The HTRF Total ALK kit efficiently detected ALK in various cell lines expressing WT ALK (e.g. IMR-32 cells), mutated ALK (eg SK-H-SH cells), or NPM-ALK fusion protein (e.g. SU-DHL-1 cells).

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 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.