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>
	<li>Lemmon, M. A., Schlessinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Signaling+by+Receptor+Tyrosine+Kinases.">Cell Signaling by Receptor Tyrosine Kinases.</a> Cell. 141 (7), 1117-1134 (2010).</li><li>Seet, B. T., Dikic, I., Zhou, M. M., Pawson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reading+protein+modifications+with+interaction+domains.">Reading protein modifications with interaction domains.</a> Nature Reviews Molecular Cell Biology. 7 (7), 473-483 (2006).</li><li>Coba, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotransmitters+drive+combinatorial+multistate+postsynaptic+density+networks.">Neurotransmitters drive combinatorial multistate postsynaptic density networks.</a> Science Signaling. 2 (68), (2009).</li><li>Gibson, S. K., Parkes, J. H., Liebman, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+modulates+the+affinity+of+light-activated+rhodopsin+for+g+protein+and+arrestin.">Phosphorylation modulates the affinity of light-activated rhodopsin for g protein and arrestin.</a> Biochemistry. 39 (19), 5738-5749 (2000).</li><li>Stites, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+mechanistic+models+to+integrate+and+analyze+multiple+proteomic+datasets.">Use of mechanistic models to integrate and analyze multiple proteomic datasets.</a> Biophysical Journal. 108 (7), 1819-1829 (2015).</li><li>Hause, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+binary+interaction+mapping+of+SH2+domains+via+fluorescence+polarization+reveals+novel+functional+diversification+of+ErbB+receptors.">Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional diversification of ErbB receptors.</a> PLOS ONE. 7 (9), 44471 (2012).</li><li>Lau, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+encoding+of+the+effect+of+a+partial+agonist+on+individual+opioid+receptors+by+multisite+phosphorylation+and+threshold+detection.">Quantitative encoding of the effect of a partial agonist on individual opioid receptors by multisite phosphorylation and threshold detection.</a> Science Signaling. 4 (185), (2011).</li><li>Mishra, M., Tiwari, S., Gomes, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+purification+and+analysis:+next+generation+Western+blotting+techniques.">Protein purification and analysis: next generation Western blotting techniques.</a> Expert Review of Proteomics. 14 (11), 1037-1053 (2017).</li><li>Brunner, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarking+multiple+fragmentation+methods+on+an+orbitrap+fusion+for+top-down+phospho-proteoform+characterization.">Benchmarking multiple fragmentation methods on an orbitrap fusion for top-down phospho-proteoform characterization.</a> Analytical Chemistry. 87 (8), 4152-4158 (2015).</li><li>Swaney, D. L., Wenger, C. D., Coon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Value+of+using+multiple+proteases+for+large-scale+mass+spectrometry-based+proteomics.">Value of using multiple proteases for large-scale mass spectrometry-based proteomics.</a> Journal of Proteome Research. 9 (3), 1323-1329 (2010).</li><li>Curran, T. G., Zhang, Y., Ma, D. J., Sarkaria, J. N., White, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MARQUIS:+A+multiplex+method+for+absolute+quantification+of+peptides+and+posttranslational+modifications.">MARQUIS: A multiplex method for absolute quantification of peptides and posttranslational modifications.</a> Nature Communications. 6 (1), 1-11 (2015).</li><li>Jain, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+cellular+protein+complexes+using+single-molecule+pull-down.">Probing cellular protein complexes using single-molecule pull-down.</a> Nature. 473 (7348), 484-488 (2011).</li><li>Jain, A., Liu, R., Xiang, Y. K., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+pull-down+for+studying+protein+interactions.">Single-molecule pull-down for studying protein interactions.</a> Nature Protocols. 7 (3), 445-452 (2012).</li><li>Kim, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pairwise+detection+of+site-specific+receptor+phosphorylations+using+single-molecule+blotting.">Pairwise detection of site-specific receptor phosphorylations using single-molecule blotting.</a> Nature Communications. 7 (1), 1-10 (2016).</li><li>Salazar-Cavazos, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisite+EGFR+phosphorylation+is+regulated+by+adaptor+protein+abundances+and+dimer+lifetimes.">Multisite EGFR phosphorylation is regulated by adaptor protein abundances and dimer lifetimes.</a> Molecular Biology of the Cell. 31 (7), 695 (2020).</li><li>Huyer, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+inhibition+of+protein-tyrosine+phosphatases+by+vanadate+and+pervanadate.">Mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate.</a> Journal of Biological Chemistry. 272 (2), 843-851 (1997).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Analytical Biochemistry. 150 (1), 76-85 (1985).</li><li>Keller, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Objective+lenses+for+confocal+microscopy.">Objective lenses for confocal microscopy.</a> Handbook of Biological Confocal Microscopy. , 145-161 (2006).</li><li>Hendriks, C. L. L., van Vliet, L. J., Rieger, B., van Kempen, G. M. P., van Ginkel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dipimage: a scientific image processing toolbox for MATLAB. , (1999).</li><li>fitgeotrans: Fit geometric transformation to control point pairs. The MathWorks Inc Available from: <a target="_blank" href="https://www.mathworks.com/images/ref/fitgeotrans.html">https://www.mathworks.com/images/ref/fitgeotrans.html</a> (2013)</li><li>Raghavachari, N., Bao, Y. P., Li, G., Xie, X., M&#252;ller, U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+autofluorescence+on+DNA+microarrays+and+slide+surfaces+by+treatment+with+sodium+borohydride.">Reduction of autofluorescence on DNA microarrays and slide surfaces by treatment with sodium borohydride.</a> Analytical Biochemistry. 312 (2), 101-105 (2003).</li><li>Wang, X., Park, S., Zeng, L., Jain, A., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+single-cell+single-molecule+pull-down.">Toward single-cell single-molecule pull-down.</a> Biophysical Journal. 115 (2), 283-288 (2018).</li><li>Chandradoss, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+passivation+for+single-molecule+protein+studies.">Surface passivation for single-molecule protein studies.</a> Journal of Visualized Experiments. (86), e50549 (2014).</li></ol>

The authors have nothing to disclose.

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.

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Research

JoVE Journal

Biochemistry

2.7K visualizações.  University of New Mexico School of Medicine. Este protocolo SiMPull permite uma quantificação da fosforilação proteica melhorando as condições de rotulagem e fixação de anticorpos, reduzindo a autofluorescência encontrada no canal verde e fornecendo algoritmos robustos de análise de moléculas únicas. A principal vantagem do SiMPull é que ele nos permite interrogar o estado de fosforilação de proteínas intactas individuais. Com este método, podemos obter novas informações que não são acessíveis através de técnicas...