Time-Resolved Forster Resonance Energy Transfer for Monitoring Protein Phosphorylation in Cells

The phosphorylation of certain cellular proteins is a key regulatory mechanism that leads to their activation or deactivation.

To monitor specific protein phosphorylation, take a culture plate containing adherent cells. Treat the cells with a stimulator to promote the phosphorylation of the proteins in cells. Aspirate the spent medium, and add a cell lysis buffer and a phosphatase inhibitor cocktail.

The buffer components cause cell lysis, releasing the cellular contents along with phosphorylated and non-phosphorylated proteins. The inhibitor cocktail inactivates cellular phosphatase, so the phosphorylated proteins remain intact.

Transfer the cell lysate into a low-volume detection plate. Treat the lysate with the donor and acceptor antibodies attached to different light-sensitive fluorophores.

During incubation, the donor antibodies recognize a specific epitope on the test protein, allowing them to bind the phosphorylated and non-phosphorylated variants of the protein. In contrast, the acceptor antibodies exclusively bind to the phosphorylated form of the test protein.

This binding brings the acceptor fluorophore near the donor fluorophore in the phosphorylated protein variants. This proximity triggers Förster resonance energy transfer, or FRET — a distance-dependent energy transfer phenomenon between two light-sensitive fluorophores.

Read the plate on a FRET-compatible microplate reader.

On illumination, the donor fluorophores get excited; this triggers an energy transfer to the acceptor molecules, which emit a long-lived fluorescence signal that correlates with the level of test protein phosphorylation in the cells.

To perform the assay, dispense 50 microliters of cells at the pre-optimized density, into a 96-well tissue culture-treated plate in the appropriate culture medium. Then, incubate them overnight in a 37 degrees Celsius and 5% carbon dioxide incubator.

The following day, prepare intermediate two-fold and four-fold dilution series of the test compounds, by serially diluting the compound in a serum-free medium, across 12 wells of a polypropylene 96-well plate. For cell stimulation, add 50 microliters of serum-free medium, containing the stimulator at a 2X concentration, and incubate the cells for the pre-optimized time at either room temperature or 37 degrees.

For cell inhibition, add 25 microliters of serum-free medium containing the inhibitor at a 4X concentration, and incubate the cells for the pre-optimized time at either room temperature or 37 degrees. Then, add 25 microliters of serum-free medium containing the stimulator at a 4X concentration, and incubate for the pre-optimized time. Next, to lyse the cells, repair the 1X supplemented lysis buffer, and add phosphatase inhibitor cocktail to it.

After carefully removing and discarding the cell culture medium, immediately add 50 microliters of the prepared 1X supplemented lysis buffer, and incubate for 30 minutes at room temperature, under shaking with moderate agitation.

For TR-FRET detection, prepare the 4X antibody detection mix in 1X detection buffer. Then, prepare the Eu-Ab1 and FR-Ab2 antibody solutions, as well as the Eu-Ab3 and FR-Ab4 antibody solutions in 1X detection buffer. Finally, mix pre-diluted Eu-Ab1 with pre-diluted FR-Ab2 for detection of phosphoprotein, and pre-diluted Eu-Ab3 and pre-diluted FR-Ab4 for detection of total protein.

Then, carefully pipette 15 microliters of the cell lysate from the 96-well culture plate to a well of a white low-volume 384-well microplate. Next, add 15 microliters of the positive control lysate and 15 microliters of 1X lysis buffer as a negative control, to separate assay wells.

To wells containing 15 microliters of the lysate, add 5 microliters of the corresponding 4X antibody detection mix to detect the phosphoprotein or total protein. After covering the plate with a plate sealer, incubate it at room temperature for 1 hour up to overnight depending on the assay kit.

After the incubation is complete, remove the adhesive plate sealer, and read the plate on a TR-FRET-compatible microplate reader.