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

1. Functionalization of the array with the biotinylated antibody

1. Prepare the following solutions.
   1. T50 Buffer, a solution of 10 mM Tris-HCl (pH 8.0) and 50 mM NaCl. The solution is stable for 1 month at RT.
   2. T50-BSA by supplementing T50 buffer with 0.1 mg/mL of BSA. Keep the prepared solution on ice.
   3. 10 mg/mL of sodium borohydride (NaBH₄) in 1x PBS. Prepare this immediately before use.
   4. 0.2 mg/mL of NeutrAvidin (see Table of Materials) in T50 buffer.
      CAUTION: NaBH₄ is a reducing agent and is flammable. Always purge the container with nitrogen gas after use and store it in a desiccator.
2. Functionalize the array with the biotinylated antibody.
   1. Remove the PEG-biotin functionalized arrays from the freezer and equilibrate the conical tube to RT before opening. Place the coverslip with the array oriented up onto a sealing film-lined 100 mm tissue culture (TC100) dish.
      NOTE: Minimize overhead lighting. All solutions should "bead up" on the squares defined by the hydrophobic array. Add an appropriate volume of solution to completely cover each square (typically 10-15 µL) and do not allow liquid to overflow the defined space. To rapidly remove liquids, use an in-house vacuum line attached to a vacuum flask to capture waste. Allow NaBH₄ to degas for 1 h before disposal by leaving the tube open in the chemical fume hood. NaBH₄ treatment is necessary to reduce background autofluorescence, thereby reducing false-positive single-molecule detections.
   2. Treat each square of the array with 10 mg/mL of NaBH₄ in 1x PBS for 4 min at RT. Wash three times with PBS.
   3. Incubate each square for 5 min with 0.2 mg/mL of NeutrAvidin in T50. Wash three times with T50-BSA.
      NOTE: NeutrAvidin binds to PEG-biotin and provides a binding site for biotinylated antibodies.
   4. Incubate each square for 10 min with 2 µg/mL of biotinylated POI-specific antibody in T50-BSA; wash three times with T50-BSA.
      NOTE: The present protocol uses biotinylated anti-EGFR IgG (see Table of Materials) to capture EGFR-GFP.

2. SiMPull of POI from whole-cell lysates

NOTE: Place the TC100 dish of functionalized SiMPull arrays on ice for the remainder of the SiMPull preparation. This step is the pull-down of a POI from total protein lysate. The lysate must not be reused after thawing.

1. Prepare the following solutions.
   1. Prepare 4% paraformaldehyde (PFA)/0.1% glutaraldehyde (GA) in 1x PBS.
      CAUTION: PFA and GA are toxic chemical fixatives and potential carcinogens. Wear PPE. Dispose of the chemicals as hazardous waste according to local regulations and guidelines.
   2. Prepare 10 mM Tris-HCl, pH 7.4.
2. Thaw and mix the lysate by gently pipetting up and down. Keep on ice.
3. Dilute 1 µL of the lysate into 100 µL ice-cold T50-BSA/PPI.
   NOTE: If needed, determine the appropriate dilution factor of the total protein lysate by applying a range of dilutions to the array. The optimal density of SiMPull receptors per array area is 0.04-0.08/µm².
4. Incubate the lysate on the array for 10 min; then wash four times with ice-cold T50-BSA/PPI.
5. Dilute AF647-conjugated anti-phosphotyrosine antibody (see Table of Materials) in ice-cold T50-BSA/PPI and incubate on the array for 1 h.
   NOTE: In the present protocol, a pan anti-pTyr (PY99)-AF647 IgG is used to identify the phosphorylated population of EGFR-GFP. The use of directly labeled antibodies removes the need for secondary antibodies, increasing the labeling options and improving the consistency of the results. Fluorescently-labeled antibodies can be obtained from commercial sources. If not commercially available, antibodies can be custom-labeled using standard bioconjugation techniques, and commercial bioconjugation kits are available. Each batch of fluorescently labeled antibodies needs to be tested for optimal labeling conditions by performing SiMPull to measure a dose curve and find the saturation point.
6. Wash six times with ice-cold T50-BSA for a total of 6-8 min.
7. Wash twice with ice-cold 1x PBS.
8. Incubate the array with 4% PFA/0.1% GA solution for 10 min to prevent antibody dissociation.
9. Wash twice for 5 min each with 10 mM Tris-HCl, pH 7.4/PBS to inactivate the fixatives.
   NOTE: For experiments using more than one antibody, (e.g., detecting multiple phosphotyrosine sites), repeat steps 2.5-2.9.

3. Image acquisition

NOTE: Single-molecule image acquisition is performed using a 150x TIRF objective and an image splitter that captures each spectral channel in a specific quadrant of the emCCD camera (see Table of Materials). Calibration images are first acquired to allow for channel registration and camera gain calibration with a nanopatterned channel alignment grid (nanogrid) that contains 20 x 20 arrays of 200 ± 50 nm holes at an intrahole distance of 3 ± 1 µm (total size ~60 µm × 60 µm).

1. Perform channel registration following the steps below.
   NOTE: Accurate channel registration is needed to properly calculate the colocalization of emitters. This step is critical.
   1. Clean the oil objective and 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.
      NOTE: Images with the nanogrid are acquired using transmitted light, which passes through the nanogrid and is detected in all spectral channels. Alternatively, one can use multifluorescent beads that emit fluorescence detected in each channel. Image acquisition will need to be optimized according to each microscope setup.
   2. Acquire a series of 20 images of the grid. Ensure that pixels are not saturated. Save the image series as "Fiducial."
   3. Defocus the nanogrid to create an Airy pattern. Acquire a series of 20 images for gain calibrations. Save the image as "Gain".
   4. Acquire a series of 20 images for camera offset by blocking all light from going to the camera. Save the image as "Background".
2. Acquire SiMPull images.
   NOTE: Before imaging the coverslip array, exchange the Tris solution for T50-BSA and equilibrate the array to RT.
   1. Clean the oil objective and deposit additional oil on the objective. Secure the coverslip array on the microscope stage.
   2. Optimize each fluorophore's excitation power, TIRF angle, and camera integration time. The goal is to achieve the highest signal-to-noise while minimizing the photobleaching of the sample. Record the laser power for consistency in future measurements.
      NOTE: The present study used 300 ms exposure time for the far-red channel and 1 s for the green channel. The 642 nm laser was used at approximately 500 µW laser power, while the 488 nm laser was used at 860 µW, measured before the tube lens.
   3. Acquire images for each sample. Image the far-red channel first, followed by each lower wavelength fluorophore to reduce photobleaching. Due to the low volume used for each sample, check the buffer level every 30-45 min and replenish as needed.