Phospho-TFEB (Ser211) assay principle

The Phospho-TFEB (Ser211) assay measures TFEB when phosphorylated at Ser211. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay 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-TEFB (Ser211) 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-TFEB (Ser211) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-TFEB (Ser211) one-plate assay protocol

Detection of Phosphorylated TFEB (Ser211) 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.

Phospho-TFEB (Ser211) detection on various human cell lines

Phospho-TFEB (Ser211) expression levels were assessed with the HTRF Phospho-TFEB (Ser211) kit in different cell lines: THP1, HeLa, HEK293T, and U87-MG.

These 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 3 mL of supplemented lysis buffer #4 (1X) for 30 minutes 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-TFEB (Ser211) detection reagents.

HTRF Phospho-TFEB (S211) assay compared to Western Blot

THP1 cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After a 48h incubation, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1X) 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-TFEB (S211) 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-TFEB (S211) assay is 8 times more sensitive than the Western Blot technique.

TFEB (transcriptional factor EB) is a transcription factor which belongs to the MiTF/TFE family. TFEB induces transcription of the genes encoding proteins associated with autophagy and lysosomal biogenesis, via a consensus sequence encountered in more than 96 lysosomal gene promoters (CLEAR motif - Coordinated Lysosomal Expression And Regulation).

TFEB activity is controlled by phosphorylation events which are mediated by different kinases, including AKT, mTOR, and ERK2.

Under nutrient rich conditions, TFEB is phosphorylated by mTOR on Ser 211 and sequestered into the cytosol, whereas under starvation or lysosomal dysfunction, TFEB is dephosphorylated and translocated into the nucleus to promote gene transcription.

TFEB acts as an upstream regulator of macroautophagy, which is a physiological process maintaining cellular homeostasis. It makes TFEB a new therapeutic target for various disorders  (ageing related diseases, infectious disease, lysosomal storage disorders, neurodegenerative diseases, or cancers).