Encyclopedia of Experiments: Biology
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Transcript
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.
