This work was supported by a grant from the NIH. The assays shown in Figure 1 was performed by Dr. Joseph Fasolo. We thank Dr. Rui Chen for helpful comments.

1. Probe Preparation
<ol>
	<li>Culture and purify the V5-fusion kinase probes used to examine interactions with other proteins as follows:
	<ol>
		<li>Use freshly streaked yeast strain Y258 (MATa pep4-3, his4-580, ura3-53, leu2-3,112) containing V5-fusion protein (expressed from GATEWAY vector pYES-DEST52). Use the isolated protein as probe on the microarrays. Plate the yeast on synthetic complete-uracil (Sc-Ura)/2% Dextrose/Agar and grow at 30&#160;&#176;C&#160;for 3 days from frozen culture (-80 &#176;C glycerol stock).</li>
		<li>Inoculate starter cultures (5&#8211;20 ml) from a single colony and grow overnight in Sc-Ura/2% Dextrose on shaking platform (220-250 rpm) or wheel at 30&#160;&#176;C.</li>
		<li>The following morning, inoculate 400 ml&#160;of the Sc-Ura/2% raffinose culture with sufficient starter culture to a final OD600 of 0.1.</li>
		<li>Grow the inoculums to OD600 of 0.6 followed by galactose induction of the V5-kinase fusion construct expression by adding a solution of 3x&#160;Yeast Extract/Peptone (YEP) supplemented with 6% Galactose, by adding enough to dilute the induction media by a factor of 3 so that final concentration of galactose is 2%.</li>
		<li>Induce cells at 30 &#176;C for 6 hr&#160;on a shaking platform. Use a 2 L&#160;Erlenmeyer flask to ensure appropriate aeration.</li>
		<li>Harvest cells using a JA-10 (or comparable) rotor by spinning 400 ml&#160;of cell suspension at 1,000 x g for 5 min at 4 &#176;C.</li>
		<li>Wash the cells once with 50 ml&#160;of ice cold PBS buffer&#160;and transfer to a 50 ml&#160;conical tube. Wash the pellet again in ice cold PBS buffer (without detergents or other additives) used for lysis and transfer to 2 ml&#160;snap-cap tubes for lysis.</li>
		<li>Spin the cells at 20,000 x g for 1 min at 4 &#176;C to a pellet and pipette away the buffer.</li>
		<li>Place tubes on ice and proceed with lysis step.</li>
		<li>Lyse the cells (250-350 &#181;l pellet) with 0.5 mm zirconia beads in a 1:1:1 volume of cell pellet, beads, and Phosphate-buffered saline ( PBS) lysis buffer and vortex the mixture using a agitation platform 3 times at 2 min intervals at 4 &#176;C.</li>
		<li>Centrifuge the lysate at 20,000 x g in a tabletop microfuge for 10 min at 4 &#176;C.</li>
		<li>Pipette supernatant into polycarbonate high speed centrifuge tube and clarify by ulracentrufugation for 30 min at 150,000 x g at 4 &#176;C.</li>
		<li>Transfer the clarified lysate to a tube containing prewashed Ni2+ affinity resin (~100 &#181;l) and incubate on a nutator for 2 hr&#160;at 4 &#176;C to capture Histidine(His)6X tagged V5-fusion protein.</li>
		<li>Wash the resin three times for 10 min&#160;at 4 &#176;C with Wash Buffer. Pellet the resin using a table top centrifuge for 5 min at 1,000 x g at 4 &#176;C and aspirate the supernatant. Add fresh wash buffer for each wash and return the tube the nutator for agitation during the wash.</li>
		<li>After the wash steps are complete, apply the washed resin to a fresh G-25 column for elution.</li>
		<li>Apply 25 &#181;l of elution buffer in a step-wise manner beginning with 100 mM Imidazole to 500 mM imidazole in 50 mM increments for a total of 9 fractions.</li>
		<li>Assay the collected fractions using sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and coomassie staining to identify the most highly enriched fraction(s) for use in the assay and add glycerol to 30% and store at -80&#160;&#176;C&#160;until used in the assay.</li>
	</ol>
	</li>
</ol>
2. Probing the Arrays
NOTE: Please see step 2.6 of this section to prepare the antibody solution before beginning the assay.
<ol>
	<li>To detect interactions, dilute the V5-fluorophore conjugated antibody (i.e. AlexaFluor 647 ) to 260 ng/ml&#160;in probe buffer and mix thoroughly by shaking. 
	NOTE: Prepare this antibody solution 30 min&#160;prior to use and place the tube on a nutator or wheel to ensure complete mixing and a homogenous suspension of antibody.</li>
	<li>Dilute the V5-fusion protein probe over a concentration range of 5&#8211;500 &#956;g/ml. 
	NOTE: Optimized for each protein&#8211;protein interaction assay, using probe buffer. Optimization involves adding more probes to probe buffer. Typically 10 &#181;g/ml is used as starting point and adjusted accordingly based on the signal strength.</li>
	<li>Remove the protein microarrays from the freezer (&#8722;20 &#176;C) and bring to 4 &#176;C in the refrigerator just prior to use.</li>
	<li>Add blocking buffer directly to the slide holder containing the protein micorarrays and cover the top with parafilm to prevent leakage. Block the arrays in blocking buffer for 1 hr&#160;by shaking at 50 rpm on a stage at 4 &#176;C.</li>
	<li>After blocking, transfer the arrays to a humidified chamber chilled to 4&#160;&#176;C, and add 90 &#956;l&#160;of diluted probe directly to the array surface. Overlay the arrays with a raised lifter slip and incubate static (no shaking) in the humidified chamber at 4&#160;&#176;C&#160;for 1.5 hr.</li>
	<li>Wash the arrays 3 times for 1 min each in probe buffer in three 50 ml&#160;conical tubes. Add the slide to conical tubes containing enough pre-chilled probe buffer to completely envelope the slide. Allow the lifter slip to gently slide off of the protein microarray (do not force it off as this could result in damage to the array surface).</li>
	<li>Apply the antibody solution directly to array immediately after completing the wash (step 2.5) and overlay with a raised lifter slip as before. Incubate the arrays for 30 min at 4 &#176;C in the humidified chamber.</li>
	<li>Perform the same wash step as before (3 times 1 min in probe buffer), and spin in a 50 ml&#160;conical tube at 800 x g in a tabletop centrifuge for 5 min at room temperature. Air-dry the arrays in a slide holder in the dark for 30 min prior to scanning the array at 647 nm.</li>
</ol>

<ol>
	<li>Fasolo, 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=Diverse+protein+kinase+interactions+identified+by+protein+microarrays+reveal+novel+connections+between+cellular+processes.">Diverse protein kinase interactions identified by protein microarrays reveal novel connections between cellular processes.</a> Genes. 25, 767-778 (2011).</li><li>Zhu, X., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProCAT:+a+data+analysis+approach+for+protein+microarrays.">ProCAT: a data analysis approach for protein microarrays.</a> Genome biology. 7, R110 (2006).</li><li>Breitkreutz, 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=A+global+protein+kinase+and+phosphatase+interaction+network+in+yeast.">A global protein kinase and phosphatase interaction network in yeast.</a> Science. 328, 1043-1046 (2010).</li><li>Gavin, A. 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=Functional+organization+of+the+yeast+proteome+by+systematic+analysis+of+protein+complexes.">Functional organization of the yeast proteome by systematic analysis of protein complexes.</a> Nature. 415, 141-147 (2002).</li><li>Jeong, J. S., 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=Rapid+identification+of+monospecific+monoclonal+antibodies+using+a+human+proteome+microarray.">Rapid identification of monospecific monoclonal antibodies using a human proteome microarray.</a> Mol Cell Proteomics. 11 (6), (2012).</li><li>Krogan, N. 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=Global+landscape+of+protein+complexes+in+the+yeast+Saccharomyces+cerevisiae.">Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.</a> Nature. 440, 637-643 (2006).</li><li>Popescu, S. 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=Differential+binding+of+calmodulin-related+proteins+to+their+targets+revealed+through+high-density+Arabidopsis+protein+microarrays.">Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 4730-4735 (2007).</li><li>Zhu, H., 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=Global+analysis+of+protein+activities+using+proteome+chips.">Global analysis of protein activities using proteome chips.</a> Science. 293, 2101-2105 (2001).</li><li>Ptacek, 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=Global+analysis+of+protein+phosphorylation+in+yeast.">Global analysis of protein phosphorylation in yeast.</a> Nature. 438, 679-684 (2005).</li><li>Haab, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+arrays+in+cancer+research.+Molecular+&amp;+cellular+proteomics.">Antibody arrays in cancer research. Molecular &amp; cellular proteomics.</a> Mol Cell Proteomics. 4 (4), 377-383 (2005).</li><li>Kopf, E., Zharhary, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+arrays--an+emerging+tool+in+cancer+proteomics.">Antibody arrays--an emerging tool in cancer proteomics.</a> The international journal of biochemistry &amp; cell biology. 39, 1305-1317 (2007).</li><li>Berrade, L., Garcia, A. E., Camarero, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+microarrays:+novel+developments+and+applications.">Protein microarrays: novel developments and applications.</a> Pharmaceutical research. 28, 1480-1499 (2011).</li><li>Balboni, I., Limb, C., Tenenbaum, J. D., Utz, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+microarray+surfaces+and+arraying+parameters+for+autoantibody+profiling.">Evaluation of microarray surfaces and arraying parameters for autoantibody profiling.</a> Proteomics. 8, 3443-3449 (2008).</li><li>Gelperin, D. 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=Biochemical+and+genetic+analysis+of+the+yeast+proteome+with+a+movable+ORF+collection.">Biochemical and genetic analysis of the yeast proteome with a movable ORF collection.</a> Genes &amp; development. 19, 2816-2826 (2005).</li><li>Fasolo, J., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+microarrays.">Protein microarrays.</a> Methods Mol. Biol. 548, 209-222 (2009).</li><li>Im, H., Snyder, M., Coligan, J. 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=Preparation+of+recombinant+protein+spotted+arrays+for+proteome-wide+identification+of+kinase+targets.">Preparation of recombinant protein spotted arrays for proteome-wide identification of kinase targets.</a> Current protocols in protein science. 27, Unit 27 24 (2013).</li><li>MacBeath, G., Schreiber, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printing+proteins+as+microarrays+for+high-throughput+function+determination.">Printing proteins as microarrays for high-throughput function determination.</a> Science. 289, 1760-1763 (2000).</li></ol>

The authors declare that they have no competing financial interests.

The protocol presented was originally performed using 85 unique yeast protein kinase-V5 fusion proteins to compare binding activity across distinct and related families of yeast protein kinases resulting in the identification of new kinase interaction networks in vivo1. As an emerging proteomic profiling technology, the development of High Throughput (HTP) screening of proteomes using protein microarrays relied on peptide libraries, and eukaryotic and prokaryotic model organisms8,17; later, this assay platform was applied to higher eukaryotes such as plant and human5,7. Currently there is no commercially available yeast protein array. However, sequence verified cDNA collections are available from multiple resources
Although the technique can be powerful, one caveat to keep in mind is that the assay is done in vitro, and, thus results will have false positives. Thus, optimizing assay condition and strategic filtering scheme is advisable to retrieve meaningful data. Next generation protein microarray technologies contain a much larger number of highly purified functional proteins for biochemical analysis across a multitude of organisms including A. thaliana, S. cerevisiae, and Homo Sapien.
The level of sophistication that the new protein microarrays have engineered into them enables an expansive variety of biochemical analyses that can be performed. Nearly the entire proteome has been cloned and purified for S. cerevisiae using a C-terminal tagged library to ensure proper post-translational modification of the proteins14. Moreover, the rapid parallel analyses offered using protein microarrays enables an unbiased approach to protein-protein interaction and post-translational modification profiling.

The need to perform global analyses of protein biochemistry and binding activity in vivo has resulted in the development of new methods for profiling protein-protein interactions (PPIs) and the post-translational modifications of whole proteomes1,3-8. Protein microarrays are manufactured as functional protein microarrays using full-length functional proteins4-6,8,9, or analytical protein microarrays containing antibodies10,11. They are engineered to contain a high-density of proteins arrayed onto microscope slides with a variety of surface chemistries to facilitate a variety of experimental conditions required for conducting wide-ranging biochemical analyses12. Nitrocellulose and aldehyde surface chemistries for chemical attachment through lysine or affinity attachment methods such as nickel- chelated slides for attaching His-tagged proteins and glutathione for affinity attachment among others13.
The use of functional protein microarrays to detect protein-protein interactions requires access to a high-quality functional protein library14. S. cerevisiae is amenable to producing such a library through the pairing of high-copy affinity tagged protein constructs with high-throughput chromatographic purification techniques. The vast majority of the yeast genome has been sequenced and nearly the entire proteome can be expressed from a high-copy plasmid for purification and biochemical analyses12. Once the proteins are obtained and arrayed in 384-well format, they are printed onto a microscope slide allowing for rapid parallel multi-parametric biochemical analysis and bioinformatic interrogation8,14-16. Protein microarrays have been used for enzymatic assays and interactions with proteins, lipids, small molecules, and nucleic acids among many other applications. The accessibility of proteins on the surface of proteome arrays make them amenable to different types of analytical detection including, immune-affinity, Surface Plasmon Resonance, fluorescence and many other techniques. Moreover, it allows for fine control of the experimental condition where it might be hard to do in vivo.
The aim of this protocol is to demonstrate the appropriate use of functional protein microarrays to detect protein-protein interactions. This application enables the high-throughput parallel biochemical analysis of protein binding activities using a highly purified analyte (protein) of interest. A C-terminal(carboxy-terminal) tagged V5-fusion bait protein of interest is produced from a high-copy plasmid in a yeast strain optimized for protein purification. C-terminal tagging ensures that the full-length protein has been translated. The protein used in this study is Tda1-V5 fusion protein kinase, which is purified using nickel affinity resin via a His6X tag. The Tda1-V5 fusion construct is purified through serial elution using an imidazole gradient to elute the most highly enriched fraction for use in the assay.

<table><tbody><tr><td>Petri Dishes</td><td>VWR</td><td>NC-10747</td><td>or comparable</td></tr><tr><td>Bacto yeast extract</td><td>Difco</td><td>0217-17</td><td></td></tr><tr><td>Bacto peptone</td><td>Difco</td><td>0118-17</td><td></td></tr><tr><td>Bacto agar</td><td>Difco</td><td>0140-01</td><td></td></tr><tr><td>Dextrose</td><td>Sigma</td><td>D9434</td><td></td></tr><tr><td>Raffinose</td><td>Sigma</td><td>R0514</td><td></td></tr><tr><td>Galactose</td><td>Sigma</td><td>G0750</td><td></td></tr><tr><td>Sc-Ura drop out media</td><td>MP Bio</td><td>114410622</td><td></td></tr><tr><td>Small Culture tubes</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>Large Erlenmeyer Flask</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>Centrifuge JA-10 rotor</td><td></td><td></td><td></td></tr><tr><td>Centrifuge tubes (500 ml)</td><td></td><td></td><td></td></tr><tr><td>Falcon tube (50 ml)</td><td>VWR/Fisher</td><td>21008-951</td><td></td></tr><tr><td>PBS Tablets</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>FastPrep Tubes (2 ml screw cap tube)</td><td></td><td></td><td></td></tr><tr><td>FastPrep Machine</td><td>MP Biomedicals</td><td>116004500</td><td></td></tr><tr><td>Zircon Beads</td><td>BioSpec</td><td>11079105</td><td></td></tr><tr><td>Triton X100</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D9779</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S3014&amp;nbsp;</td><td></td></tr><tr><td>Imidazole (pH = 7.4)</td><td>Sigma</td><td>I5513&amp;nbsp;</td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td>93482</td><td></td></tr><tr><td>Complete Inhibitor Cocktail (EDTA Free)</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>Phosphatase inhibitor cocktail 1</td><td>sigma</td><td>P2850</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153&amp;nbsp;</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P1379&amp;nbsp;</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A1852&amp;nbsp;</td><td></td></tr><tr><td>Shaking Incubator (30 &amp;deg;C)</td><td></td><td></td><td></td></tr><tr><td>Nickel Affinity Resin</td><td>Life Technologies</td><td>R901-01</td><td></td></tr><tr><td>G25 column</td><td>GE life sciences</td><td>27-5325-01&amp;nbsp;</td><td></td></tr><tr><td>Nutator</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>SDS-PAGE Gel (NuPage)</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Coomassie Blue Stain</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Monoclonal V5 Antibody</td><td>Life Technologies</td><td>R960-25</td><td></td></tr><tr><td>Alexa647 Tagged monoclonal V5 Antibody</td><td>Life Technologies</td><td>451098</td><td></td></tr><tr><td>Protein Microarrays Kit</td><td>Life Technologies</td><td>PAH0525013</td><td></td></tr><tr><td>Genepix Scanner</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Lifter Slips</td><td>Thomas Scientific</td><td></td><td>http://www.thomassci.com/Supplies/Microscope-Cover-Glass/_/LIFTER-SLIPS/</td></tr><tr><td>Lysis Buffer: 0.1% Triton X-100, 0.5 mM DTT,&lt;br/&gt; 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 500 mM NaCl, 50 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &amp;ndash; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF&amp;nbsp; (add just prior to use)</td><td></td><td></td><td>Add the reagents to 1x PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride; pH 7.4) to obtain the desired volume of lysis solution</td></tr><tr><td>Blocking Buffer: 1% bovine serum albumin (BSA), 0.1% Tween-20</td><td></td><td></td><td>Add the reagents to 1x PBS to obtain the desired volume of blocking buffer</td></tr><tr><td>Probe Buffer: 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 0.5 mM DTT, 0.05% Triton X-100, 50 mM NaCl, 500 &amp;mu;M ATP (for kinases), 1% BSA</td><td></td><td></td><td>Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>Wash Buffer: 0.1% Triton X-100, 500 mM NaCl, 0.5 mM DTT, 50 mM imidazole (pH 7.4),2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>Elution Buffer: 0.1% Triton X-100, 0.5 mM DTT, 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 500 mM NaCl, 50 &amp;ndash; 500 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &amp;ndash; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF (add just prior to use)</td><td></td><td></td><td>Before you begin, it is important to note the concentration gradient of imidazole (50 &amp;ndash; 500 mM) and to create separate tubes for each concentration (it is advisable to create the interval using 50 mM increments). Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>3x YEP +6% galactose: Dissolve 30 g of yeast extract and&amp;nbsp; 60 g of peptone in 700 ml of H2O, and autoclave. Add 300 ml of filter sterilized 20% galactose (wt/vol)</td><td></td><td></td><td>&amp;nbsp;Galactose should not be autoclaved</td></tr></tbody></table>

probing high-density functional protein microarrays to detect protein-protein interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

The protein-protein interaction activity was observed using a standard chip reader to evaluate the Tda1-V5 protein kinase fusion construct as a bait protein against a yeast functional protein microarray containing approximately 4,200 unique S. cerevisiae GST-fusion proteins. Further interrogation with the Genepix software revealed a multitude of binding events of varying intensities. The affinity was gauged from the graded intensity of the signal derived from a monoclonal V5-Fluorophore conjugated antibody bound to its target (V5-kinase). ProCat scoring algorithm2 was used to identify protein-protein interactions across two separate protein microarrays for each of the proteins assayed in duplicate (each protein is spotted twice on the array), then a threshold cut-off was determined for scoring the observed interactions.
By comparing two arrays one obtains 4 technical replicates (n = 4) that can use to determine inter-assay variation through correlation (R2) of the two spots for each protein on the arrays. A total of 9 protein-protein interactions were identified between the Tda1-V5 bait protein and the proteins on the microarray using a predefined threshold for identifying unique protein kinase interactions1. Moreover, the same proteins are spotted in different positions throughout the arrays as a control to assess surface artifacts that may contribute to false positives. The comparison of multiple binding profiles of different kinase-V5 fusion proteins enables the identification of kinase specific protein-protein interactions for testing in vivo.
In this experiment we tested the binding activity of Tda1-V5 and identified several statistically significant PPIs based on ranked by p-value compared to other kinases tested. Of particular interest is Rim11, which has been identified as a phosphorylated target of Tda1 in a previous published study9. Once targets have been identified, they can be tested for biochemical and functional activity in vivo1.
<img alt="Figure 1" src="/files/ftp_upload/51872/51872fig1highres.jpg" /> 
Figure 1: Tda1-V5 compared to empty vector control. Yeast protein microarrays spotted in duplicate with ~4,200 full length GST-fusion protein was incubated with control or Tda1&#8211;V5 probes. Inset panel compares the identical region in both arrays. Protein-protein interaction was detected (blue boxes) between Rim11 and Tda1-V5.

Watch this Scientific Journal Video about Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions at JoVE.com

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

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8.0K Views.  Stanford University. Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

Video: Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Biochemistry

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Immunology and Infection

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

High-Sensitivity Detection of Compound-Protein Binding Using Fluorescence

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. Umbelliferone, an active ingredient in Tibetan medicine Vicatia thibetica, exhibits an immunomodulatory effect with an unknown mechanism. The CD40 protein is a key target in the immune response. Therefore, this study employs the principle of differential scanning fluorescence technology to analyze the interactions between CD40 protein and umbelliferone using fluorescent enzyme markers. Initially, the stability of the protein fluorescent orange dye was experimentally verified, and the optimal dilution ratio of 1:500 was determined. Subsequently, it was observed that the temperature melting (Tm) value of CD40 protein tended to decrease with an increase in concentration. Interestingly, the interaction between CD40 protein and umbelliferone was found to enhance the thermal stability of CD40 protein. This study represents the first attempt to detect the binding potential of small molecule compounds and proteins using fluorescence microplates and fluorescent dyes. The technique is characterized by high sensitivity and accuracy, promising advancements in the fields of protein stability, protein structure, and protein-ligand interactions, thus facilitating further research and exploration.

Author Spotlight: Streamlining PCR Methods for Enhanced Accessibility and Efficiency

This protocol describes a unique method for detecting the binding between compounds and protein molecules, offering the advantages of minimal protein sample loss and high data accuracy.

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. ...

Medicine

Protein Networks

An organism can have thousands of different proteins, and these proteins must cooperate to ensure the health of an organism. Proteins bind to other proteins and form complexes to carry out their functions. Many proteins interact with multiple other proteins creating a complex network of protein interactions.
These interactions can be represented through maps depicting protein-protein interaction networks, represented as nodes and edges. Nodes are circles that are representative of a protein, and edges are lines that connect two interacting proteins. These networks provide a way to visualize the complexity of the protein-protein interactions in a system. These maps can include both stable interactions, like the ones formed in protein complexes, as well as transient interactions. The protein interactions occurring in a cell, an organism, or a specific biological context can be collectively called an &#8216;interactome&#8217;.
Protein networks can be studied using various biochemical and computational methods. One of the first steps in studying protein interactions is to isolate a protein of interest along with the other associated proteins. This can be carried out by tagging the protein of interest with an affinity tag, such as a histidine tag. This tag can then be used to separate the protein along with the other proteins using affinity chromatography. Isolated proteins are then digested with a protease, such as trypsin,&#160; and then analyzed using liquid chromatography-mass spectrometry (LC-MS). The peptide mass can then be compared to a database with known protein sequences to determined its identity.
Computationally, protein-protein interactions can be analyzed using databases as well as prediction tools. There are various databases, such as IntAct managed by EMBL-EBI, that consist of experimentally validated and predicted protein interactions. Other tools like STRING by the Swiss Institute of Bioinformatics can be used to predict these interaction networks.
The study of protein networks can lead to scientific discoveries, such as determining the function of an unknown protein. Examining the changes in these networks can help elucidate the differences between healthy and diseased cells. This information can also be used for crucial applications such as designing drugs for the treatment of diseases.&#160; Analysis of protein networks can identify highly connected nodes that may be crucial for cellular survival, which can be targeted in cancer and diseases where cell death is desired but would be unsuitable for most diseases. On the other hand, less connected nodes that only interact with a few specific pathways may be targeted if a specific cell function is affected, and designing drugs that interact with these less connected nodes may lead to fewer side effects.

Protein Networks and PredictionTools: IntAct and STRING

An organism can have thousands of different proteins, and these proteins must cooperate to ensure the health of an organism. Proteins bind to other ...

Education

JoVE Core

Molecular Biology

Unit 1

Protein Function

KEY TERMS AND CONCEPTS

Fluorescence Anisotropy-Based Detection of Protein-Protein Interactions

Mix 3 nanomoles of the SBDS-FlAsH protein with 3 nanomoles of the Lumio Green dye in a 5-microliter volume of anisotropy buffer. Let the reaction proceed for 8 hours at 4 degrees Celsius. After 8 hours, dialyze the sample against the anisotropy buffer overnight to remove the free dye. Measure the absorbance at 280 nanometers and 508 nanometers in a spectrophotometer using a quartz cuvette. Then, use the Lambert-Beer law to quantify the percent of labeled protein as described in the text protocol.
Before measuring the anisotropy value, each titration step of the protein-ligand should be done carefully, ensuring that the entire sample is dispensed into the solution, and becomes homogeneous.
In a fluorescence cuvette, place 200 microliters of 30 nanomolar SBDS-FlAsH in anisotropy buffer, and titrate 2 microliters of 30 micromolar EFL1. Mix thoroughly, and let the reaction stand for 3 minutes before measuring the anisotropy and fluorescence value. Repeat this process until a total volume of 40 microliters of EFL1 has been added. As a final step, fit the data to a presumed binding model, as described in the text protocol.

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Summary

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Chapters in this video

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

Protein Networks

Fluorescence Anisotropy-Based Detection of Protein-Protein Interactions