Single-Molecule Pull-Down Assay for Protein Phosphorylation Analysis: A High Throughput Technique to Quantify Protein Phosphorylation in Cell Lysate

The phosphorylation of certain cellular proteins is important for their biological function.

To quantify the phosphorylation levels of intact proteins using a single-molecule pull-down assay, begin by taking a glass coverslip with a grid-array pattern. Each compartment is coated with biotinylated polyethylene glycol or PEG-based linkers interspaced with normal PEG to prevent steric hindrance during protein binding.

Treat the coverslip with a reducing reagent to minimize the autofluorescence in analysis. Overlay the coverslip with Neutravidins - biotin-binding proteins - and incubate, allowing them to bind biotinylated PEG linkers.

Supplement the array with specific anti-target protein antibodies. Each antibody contains biotin linked to its Fc region, which helps anchor the antibody to the neutravidin, causing immobilization.

Now, add the cell culture lysate containing various proteins, including the green fluorescence-tagged phosphorylated and non-phosphorylated variants of the target protein, and incubate. Both variants of the target protein get captured by the antibodies, forming complexes. Wash to remove the unbound proteins.

Next, treat these complexes with the second set of red fluorescence-tagged antibodies, which exclusively bind the phosphorylated residues of the protein, imparting a different fluorescence signal to the complex.

Image the array under a fluorescence microscope and observe for two distinct fluorescence signals. The colocalization of both fluorescence signals indicates the presence of phosphorylated proteins in the sample.