HTRF Phospho-ULK1 (Ser556) assay principle

The Phospho-ULK1 (Ser556) assay measures ULK1 when phosphorylated at Ser556. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. It uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both 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-ULK1 (S556) two-plate assay protocol

The 2-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 Phospho-ULK1 (Ser556) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-ULK1 (Ser556) one-plate assay protocol

Detection of Phosphorylated ULK1 (Ser556) 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 Phospho S556 ULK1 assay

Phospho S556 ULK1 expression levels were assessed with the HTRF Phospho S556 ULK1 kit in different HAP1 cell lines.

HAP1 Wild Type (HAP1 WT) and HAP1 knocked out for ULK1 (HAP1 KO ULK1) cell lines were cultured in T175 flasks in complete medium at 37°C, 5% CO2. After a 48h incubation, cells were lysed with 1mL of supplemented lysis buffer #4 (1X) for 30.10E6 cells, for 30min at RT under gentle shaking.

16µL of each lysate were transferred into a low volume white microplate before the addition of 4 µL of HTRF Phospho (S556) ULK1 detection reagents.

Versatility of Phospho-S556 ULK1 assay using various human cell lines

-Phospho-S556 ULK1 expression levels were assessed with the HTRF Phospho S556 ULK1 kit in different cell lines : U-87 MG, MCF7, HeLa.

These different cell lines were cultured in T175 flasks in complete culture medium at 37°C, 5% CO2. After a 48h incubation, the cells were lysed with 1mL of supplemented lysis buffer #4 (1X) for 30.10E6 cells, for 30min at RT under gentle shaking.

16 µL of each lysate were transferred into a low volume white microplate before the addition of 4 µL of HTRF Phospho S556 ULK1 detection reagents.

HTRF Phospho-S556 ULK1 assay compared to Western Blot

U-87 MG cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After a 48h incubation, cells were lysed with 1mL of supplemented lysis buffer #4 (1X) for 30.10E6 cells, for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Phospho S556 ULK1 detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

In these conditions, the HTRF Phospho S556 ULK1 assay is 8 times more sensitive than the Western Blot technique.

ULK1 is a cytoplasmic kinase part of the ULK1 complex comprising FIP200, Atg101, and Atg13. Activation of the ULK complex promotes the formation of the phagophore and the initiation of autophagy.

The ULK1 complex is negatively regulated by mTORC1, whereas AMPK mediates its activation. mTORC1 inhibits the ULK1 complex through direct phosphorylation of Ser758 on ULK1. Under nutrient starvation, AMPK promotes autophagy by activating ULK1 through phosphorylation at Ser317 and Ser556, or through inhibition of mTORC1.

The ULK1 complex is considered to be an upstream hub of autophagy, which is a physiological process maintaining cellular homeostasis. This makes ULK1 a therapeutic target, notably for cancer therapy. For example, ULK1 inhibition induced apoptosis in non-small cell lung cancer (NSCLC) cells.