An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

This SiMPull protocol enables a quantification of protein phosphorylation by improving antibody labeling and fixation conditions, reducing autofluorescence found in the green channel and providing robust single molecule analysis algorithms. The primary advantage of SiMPull is that it allows us to interrogate the phosphorylation status of individual intact proteins. With this method, we can gain novel information that's not accessible through traditional techniques, such as western blotting and mass spec analysis.

We have a team of researchers to help demonstrate the protocol. First, Julian Rojo will show how to piranha etch the cover glass. Rachel Grattan will then show the steps for coating of the cover glass.

And Elizabeth Bailey will show how to draw the array and image samples. To begin, piranha etch the cover slips by gently agitating the reaction beaker every five minutes for 30 minutes. Neutralized the piranha solution by gradually adding a weak base.

With a glass rod, transfer the etched cover slips into a Buchner funnel and rinse for five minutes in double distilled water. Then place the cover slips in a glass Coplin jar and cover with methanol. Seal the lid with a sealing film and bath sonicate for 10 minutes.

After sonication, empty the methanol into a glass storage bottle. Repeat this step with acetone. Rinse the cover slips thrice with double distilled water in the Coplin jar.

Drain the water from the cover slips and dry them by waving through the flame of a Bunsen burner. Place the cover slips in a dry Coplin jar. Then add amino silane solution to the Coplin jar to perform cover slip amino silanization.

Cover and apply a sealing film to protect from light. Rinse the cover slips with methanol and discard the used solution. Again, rinse the cover slips thrice with double distilled water for two minutes each.

Dab away excess moisture and air dry completely for 10 minutes. Next, draw grid array with a hydrophobic barrier pen. Mark an identifier on the cover slip to identify the proper orientation.

After the ink dries, place the cover slips in a humidified chamber. Resuspend 153 milligrams of mPEG and 3.9 milligrams of biotin-PEG in 609 microliters of 10 millimolar sodium bicarbonate and vortex. Centrifuge at 10, 000 x g from one minute at room temperature to remove bubbles.

Apply 10 to 15 microliters of this solution per square to completely cover the array without overflowing. Store the cover slips into humidity chamber in the dark for three to four hours at room temperature. Then wash the cover slips by dipping them for 10 seconds sequentially into three beakers filled with double distilled water.

Remove all moisture from the cover slips using nitrogen gas. Store the cover slips back to back in a 50 milliliter conical tube filled with nitrogen and seal with a sealing film. Wrap the tube with sealing film and place it at minus 20 degree Celsius.

Remove the biotin-PEG functionalized arrays from the freezer and equilibrate them to room temperature. Place the cover slip 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 in PBS for four minutes at room temperature.

Wash thrice with PBS. Next incubate with 0.2 milligrams per milliliter of NeutrAvidin in T50 for five minutes, followed by washing thrice with T50 BSA. Repeat the incubation with two micrograms per milliliter of biotinylated POI specific antibody in T50 BSA for 10 minutes followed by washing.

First, thaw mix the lysate by pipetting. Dilute one 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 one 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 images background. To acquire simple images, first, clean the oil objective and deposit additional oil on the objective. Secure the cover slip 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. Using this technique, EGFR-GFP was captured from total protein lysates.

The autofluorescence of the hydrophobic ink was used as a guide to finding the focal plane of the sample. Raw data images were acquired with spectral channels separated on the camera chip. An overlay of the green and far-red channels was further examined for data acquisition.

Channel registration was performed on images acquired from the nanogrid. An overlay of fiducial images showed incomplete registration. Alignment was done by applying a local weighted mean transform on the far-red and green channel coordinates, which was used to register the subsequent SiMPull data.

Single emitters above the background photon count from the GFP and AF647 channels were boxed and Gaussian localization was performed to identify the locations. Colocalization of EGFR-GFP in a F647 was done to identify phosphorylated receptors. And the percentage of colocalization was used to determine the fraction of phosphorylated receptors.

Background detections were reduced when piranha etched glass was treated with sodium borohydride. Robust binding of EGFR-GFP in the lysate was observed with minimal non-specific PY99-AF647 binding, showing retention of surface functionalization using this technique. SiMPull provides information about protein phosphorylation and can address a broad range of self-signaling questions.

This can enhance our understanding of signaling in both normal and disease states.