Total TFEB assay principle

The HTRF Total TFEB assay quantifying the expression level of TFEB is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses two antibodies, one coupled to a donor fluorophore and the other to an acceptor. The two antibodies are highly specific for a distinct epitope on the protein. In presence of TFEB 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 TFEB 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 the Total TFEB HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total TFEB one-plate assay protocol

Detection of total TFEB 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.

Total TFEB detection on various human cell lines

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

The 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 Total TFEB detection reagents.

HTRF Total TFEB 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 Total TFEB detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

In these conditions, the HTRF Total TFEB assay is 16 times more sensitive than the Western Blot technique.

TFEB Signaling Pathway

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).