Name: an optimized single-molecule pull-down assay for quantification of protein phosphorylation
Uploaded: 2022-06-06T13:00:00
Description: Watch this Scientific Journal Video about An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation at JoVE.com

This work was supported by the National Institutes of Health R35GM126934, R01AI153617, and R01CA248166 to DSL. EMB was supported through the ASERT-IRACDA program (NIH K12GM088021) and JAR by the UNM MARC program (NIH 2T34GM008751-20). We gratefully acknowledge using the University of New Mexico Comprehensive Cancer Center fluorescence microscopy shared resource, supported by NIH P30CA118100. We want to acknowledge Drs. Ankur Jain and Taekijip Ha, whose original development of SiMPull inspired this work. 
ES-C present address: Immunodynamics Group, Laboratory of Integrative Cancer Immunology, Center for Cancer Research, National Cancer Institute, Bethesda

1. Coverslip preparation
NOTE: For this step, one needs to wear personal protective equipment (PPE), which includes a double layer of nitrile gloves, safety glasses or face shield, and a lab coat.
<ol>
	<li>Perform piranha etching to remove organic debris from the glass. 
	CAUTION: Piranha solution is a strong oxidizing agent that is corrosive and highly reactive when in contact with organic materials. Reaction with organic debris is exothermic and potentially explosive...

<ol>
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The protocol described here was optimized to enable quantitative measurements of receptor phosphorylation at the single protein level. Several straightforward but important modifications to the SiMPull protocol were developed that improved the reliability of the measurement for phospho-tyrosine detection, including reduction of autofluorescence with NaBH4 treatment and postfixing of the sample to prevent antibody dissociation. Using the green channel mask to identify receptor locations for calculation of coloc...

Membrane-associated signaling is tuned by a combination of ligand-induced membrane receptor activation and recruitment of downstream accessory proteins that propagate the signal. Phosphorylation of key tyrosines in receptor cytoplasmic tails is critical to initiating the formation of signaling complexes, or signalosomes1,2. Therefore, an important question in biology is how phosphorylation patterns are created and maintained to recruit signaling partners and dictate cellular outcomes. This includes understanding the heterogeneity of receptor phosphorylation, both in abundance and in the specific phosphotyrosine patterns that can provide a means of manipulating signaling outputs by dictating the composition of the signalosome3,4,5,6,7. However, there are limitations in current methods to interrogate protein phosphorylation. Western blot analysis is excellent for describing trends of protein phosphorylation but is semi-quantitative8 and does not provide information on the heterogeneity of the system because thousands to millions of receptors are averaged together. While western blots allow probing a sample using phospho-specific antibodies to specific tyrosines, they cannot provide information on multisite phosphorylation patterns within the same protein. Quantitative phosphoproteomics report on phosphotyrosine abundance, but there are limitations to detecting multisite phosphorylation, as the residues of interest need to be located within the same peptide (typically 7-35 amino acids) that is generated by enzymatic digestion9,10,11.
To overcome the limitations mentioned above, the single-molecule pull-down (SiMPull) assay has been adapted to quantify the phosphorylation states of intact receptors at the single-molecule level. SiMPull was first demonstrated as a powerful tool for interrogating macromolecular complexes by Jain et al.12,13. In SiMPull, macromolecular complexes were immunoprecipitated (IP) on antibody-functionalized glass coverslips and then analyzed through single-molecule microscopy for protein subunit number and co-IP with complex components12. A modification by Kim et al.14, termed SiMBlot, was the first to use a variation of SiMPull to analyze phosphorylation of denatured proteins.&#160;The SiMBlot protocol relies on capturing biotinylated cell surface proteins using NeutrAvidin-coated coverslips, which are then probed for phosphorylation with phospho-specific antibody labeling14. Despite these advances, improvements were needed to make the quantification of posttranslational modification more robust and applicable to a broader range of proteins.
The present protocol describes an optimized SiMPull approach that was used to quantify phosphorylation patterns of intact epidermal growth factor receptor (EGFR) in response to a range of ligand conditions and oncogenic mutations15. While this work focuses on EGFR, this approach can be applied to any membrane receptor and cytosolic proteins of interest (POI), for which quality antibodies are available. The protocol includes steps to reduce sample autofluorescence, a sample array design that requires minimal sample volume with simultaneous preparation of up to 20 samples, and optimization of antibody labeling and fixation conditions. Data analysis algorithms have been developed for single-molecule detection and quantification of phosphorylated proteins.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>MTC Bio</td>
			<td>C2000</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Tris-HCl pH 7.4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Tris-HCl pH 8.0/ 50 mM NaCl</td>
			<td></td>
			<td></td>
			<td>T50 Buffer</td>
		</tr>
		<tr>
			<td>100 mm Tissue Culture dish</td>
			<td>CELLTREAT</td>
			<td>229620</td>
			<td>Storage of piranha etched glass/arrays</td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td></td>
			<td></td>
			<td>Functionalized Glass storage/ KOH reuse</td>
		</tr>
		<tr>
			<td>50 mM Tris-HCl pH 7.2/150 mM NaCl</td>
			<td></td>
			<td></td>
			<td>Lysis Buffer Component</td>
		</tr>
		<tr>
			<td>60 mm Tissue Culture dish</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>8% Glutaraldehyde Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>16020</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Acetone (C3H6O)</td>
			<td>Millipore Sigma</td>
			<td>270725</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 NHS Ester</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-20006</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal-Free Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Human EGFR (External Domain) &#8211; Biotin</td>
			<td>Leinco Technologies, Inc</td>
			<td>E101</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-p-Tyr Antibody (PY99) Alexa Fluor 647</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-7020 AF647</td>
			<td></td>
		</tr>
		<tr>
			<td>Bath-sonicator</td>
			<td>Branson</td>
			<td>1200</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Pierce</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-PEG</td>
			<td>Laysan Bio</td>
			<td>Biotin-PEG-SVA, MW 5,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Gold Biotechnology</td>
			<td>A-420-1</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Buchner funnel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Millipore Sigma</td>
			<td>C4901</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Cell Scraper</td>
			<td>Bioworld</td>
			<td>30900017-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Filtering Flask</td>
			<td>Fisher Scientific</td>
			<td>S15464</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin Jar</td>
			<td>WHEATON</td>
			<td>900470</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips 24 x 60 #1.5</td>
			<td>Electron Microscopy Sciences</td>
			<td>63793</td>
			<td></td>
		</tr>
		<tr>
			<td>DipImage</td>
			<td></td>
			<td></td>
			<td>https://diplib.org/</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Caisson Labs</td>
			<td>DML19-500</td>
			<td></td>
		</tr>
		<tr>
			<td>emCCD camera</td>
			<td>Andor iXon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Optima</td>
			<td>Bio-Techne</td>
			<td>S12450H</td>
			<td>Heat Inactivated</td>
		</tr>
		<tr>
			<td>Fusion 360 software</td>
			<td>Autodesk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Geneticin G418 Disulfate</td>
			<td>Caisson Labs</td>
			<td>G030-5GM</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid (CH3COOH)</td>
			<td>JT Baker</td>
			<td>JTB-9526-01</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Glass serological pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Stir Rod</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (D-(+)-Glucose)</td>
			<td>Millipore Sigma</td>
			<td>D9434</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Halt Phosphotase and Protease Inhibitor Cocktail (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>78446</td>
			<td>Lysis Buffer Component</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millipore Sigma</td>
			<td>H3375</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid (HCl)</td>
			<td>VWR</td>
			<td>BDH7204-1</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (H2O2) (3%)</td>
			<td></td>
			<td>HX0645</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (H2O2) (30%)</td>
			<td>EMD Millipore</td>
			<td>HX0635-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CA-630 (NP-40)</td>
			<td>Sigma Aldrich</td>
			<td>I8896</td>
			<td>Lysis Buffer Component</td>
		</tr>
		<tr>
			<td>ImmEdge Hydrophobic Barrier Pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersol 518F immersion oil</td>
			<td>Zeiss</td>
			<td>444960-0000-000</td>
			<td></td>
		</tr>
		<tr>
			<td>in-house vacuum line</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-164</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnessium Chloride Hexahydrate (MgCl2-6H2O)</td>
			<td>MPBio</td>
			<td>2191421</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks</td>
			<td></td>
			<td>Curve Fitting Toolbox, Parallel Computing Toolbox, and Statistics and Machine Learning toolbox</td>
		</tr>
		<tr>
			<td>Methanol (CH3OH)</td>
			<td>IBIS Scientific</td>
			<td>MX0486-1</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Milli-Q water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mix-n-Stain CF Dye Antibody Labeling Kits</td>
			<td>Biotium</td>
			<td>92245</td>
			<td>Suggested conjugation kit</td>
		</tr>
		<tr>
			<td>mPEG</td>
			<td>Laysan Bio</td>
			<td>mPEG-succinimidyl valerate, MW 5,000</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(2-aminoethyl)-3-aminopropyltrimethoxysilane</td>
			<td>UCT United Chemical</td>
			<td>A0700</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Nanogrid</td>
			<td>Miraloma Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NeutrAvidin Biotin Binding Protein</td>
			<td>Thermo Fisher Scientific</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen (compressed gas)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NVIDIA GPU with CUDA</td>
			<td></td>
			<td></td>
			<td>Look for compatibility at https://www.mathworks.com/help/parallel-computing/gpu-support-by-release.html</td>
		</tr>
		<tr>
			<td>Olympus iX71 Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M Sealing Film</td>
			<td>The Lab Depot</td>
			<td>HS234526C</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7.4</td>
			<td>Caisson Labs</td>
			<td>PBL06</td>
			<td></td>
		</tr>
		<tr>
			<td>PC-200 Analog Hot Plate</td>
			<td>Corning</td>
			<td>6795-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-163</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-EGF Receptor (Tyr1068) (1H12) Mouse mAb</td>
			<td>Cell Signaling Technology</td>
			<td>2236BF</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Millipore Sigma</td>
			<td>529552</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide (KOH)</td>
			<td>Millipore Sigma</td>
			<td>1050330500</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Premium PLA Filament, 1.75 mm diameter</td>
			<td>Raise 3D</td>
			<td>PMS:2035U/RAL:3028</td>
			<td>Printing temperature range: 205-235 &#176;C</td>
		</tr>
		<tr>
			<td>Pro2 3D printer</td>
			<td>Raise 3D</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex 1 L beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PYREX 100 mL storage bottles</td>
			<td>Corning</td>
			<td>1395-100</td>
			<td>CH3OH/C3H6O reuse</td>
		</tr>
		<tr>
			<td>Pyrex 250 mL beakers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex 4 L beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Quad-view Image Splitter</td>
			<td>Photometrics</td>
			<td>Model QV2</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>Eppendorf</td>
			<td>EP-5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>RevCount Cell Counters, EVE Cell Counting Slides</td>
			<td>VWR</td>
			<td>10027-446</td>
			<td></td>
		</tr>
		<tr>
			<td>Semrock emission filters: blue (445/45 nm), green (525/45 nm), red (600/37 nm), far-red (685/40 nm)</td>
			<td>Semrock</td>
			<td>LF405/488/561/635-4X4M-B-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>smite single molecule analysis package</td>
			<td></td>
			<td></td>
			<td>https://github.com/LidkeLab/smite.git</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Sigma Aldrich</td>
			<td>S6014</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Sodium Borohydride (NaBH4)</td>
			<td>Millipore Sigma</td>
			<td>452874</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Millipore Sigma</td>
			<td>S9625</td>
			<td>Activate by successive heat and pH cycling</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>VWR</td>
			<td>BDH3247-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Orthovanadate (Na3VO4)</td>
			<td>Millipore Sigma</td>
			<td>S6508</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Sulfuric Acid (H2SO4)</td>
			<td>Millipore Sigma</td>
			<td>258105</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>TetraSpeck Microspheres</td>
			<td>Thermo Fisher Scientific</td>
			<td>T7279</td>
			<td>multi-fluorescent beads</td>
		</tr>
		<tr>
			<td>Tris (Trizma) base</td>
			<td>Millipore Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain, 0.4%</td>
			<td>Thermo Fisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.05%</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300120</td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized single-molecule pull-down assay for quantification of protein phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

A cartoon depicting the SiMPull process is shown in Figure 1A. Coverslips are functionalized using NeutrAvidin as an anchor for biotinylated anti-EGFR antibodies to capture EGFR-GFP from total protein lysates. After washing away unbound protein, the phosphorylated receptors are labeled with an anti-phosphotyrosine (anti-PY) antibody15. Figure 1B shows an image of the hydrophobic array, where multiple samples can be prepared and imaged on ...

Watch this Scientific Journal Video about An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation at JoVE.com

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

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

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.

an-optimized-single-molecule-pull-down-assay-for-quantification

Research

JoVE Journal

Biochemistry

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning because it is involved in microtubule assembly and stabilization. Besides neurons, tau is expressed in human breast, prostate, gastric, colorectal, and pancreatic cancers where it shows nearly similar structure and exerts similar functions as the neuronal tau. The amount of tau and its phosphorylation can change its function as a stabilizer of microtubules, and lead to the development of paired helical filaments in different neurodegenerative disorders, such as Alzheimer&#39;s disease. Determining the phosphorylation state of tau and its microtubule-binding characteristics is important. In addition, examining the intracellular localization of tau is important in different diseases. This manuscript details standard protocols for measuring tau phosphorylation and tau binding to microtubules in colorectal cancer cells with or without curcumin and LiCl treatment. These treatments can be used to stop cancer cell proliferation and development. Intracellular localization of tau is examined by using immunohistochemistry and confocal microscopy while using low amounts of antibodies. These assays can be used repetitively for screening compounds that affect tau hyperphosphorylation or microtubule binding. Novel therapeutics used for different tauopathies or related anticancer agents can potentially be characterized using these protocols.

This manuscript describes standard protocols for measuring tau hyperphosphorylation, measuring tau binding to microtubules, and localization of intracellular tau following drug treatments. These protocols can be used repetitively for screening drugs or other compounds that target tau hyperphosphorylation or microtubule binding.

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.

The electrochemiluminescence immunoassay (ECLIA) is a novel approach for quantitative detection of endogenous and exogenously applied MeCP2 protein variants, which produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error over a wide working range. Here, the protocol for the MeCP2-ECLIA in a 96-well format is described.

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, ...