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

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.