This article describes a method for quantifying the phosphorylation levels of proteins using a single-molecule pull-down assay. The technique involves the use of a glass coverslip with a grid-array pattern and various biochemical reagents to capture and analyze phosphorylated proteins.
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.
Remove the biotin-PEG functionalized arrays from the freezer and equilibrate them to room temperature. Place the coverslip with the array facing up over a 100-millimeter tissue culture dish lined with sealing film. Incubate each square of the array with 10 milligrams per milliliter of sodium borohydride and PBS for 4 minutes at room temperature. Wash thrice with PBS.
Next, incubate with 0.2 milligrams per milliliter of NeutrAvidin in T50 for 5 minutes, followed by washing thrice with T50-BSA. Repeat the incubation with 2 micrograms per milliliter of biotinylated POI-specific antibody in T50-BSA for 10 minutes, followed by washing.
First, thaw and mix the lysate by pipetting. Dilute 1 microliter of the lysate into 100 microliters of ice-cold T50-BSA/PPI. Incubate the lysate on the array for 10 minutes, followed by washing. Prepare AF647-conjugated anti-phosphotyrosine antibody in ice-cold T50-BSA/PPI, and incubate on the array for 1 hour.
Deposit a drop of oil on the objective. Place the nanogrid on the stage for imaging. Using transmitted white light, focus on the grid pattern. Acquire a series of 20 images of the grid. Ensure that pixels are not saturated, and save the image series as "Fiducial."
Defocus the nanogrid to create an Airy pattern. Acquire a series of 20 images for gain calibrations, and save the image as "Gain." Then, acquire a series of 20 images for camera offset by blocking all light to the camera, and save the image as "Background."
To acquire SiMPull images, first, clean the oil objective, and deposit additional oil on the objective. Secure the coverslip array on the microscope stage. Acquire images for each sample, first in the far-red channel, followed by lower wavelength fluorophores. Check the buffer level every 30 to 45 minutes, and replenish as needed.
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