Name: flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction
Uploaded: 2013-08-15T12:00:00
Description: Watch this Scientific Journal Video about Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction at JoVE.com

Vi takker O&#39;Bryan laboratorium på UIC til kritisk eksperimentel evaluering og diskussion. Dette arbejde blev støttet af American Heart Association Grant 11SDG5230003 til GKC og National Center for Fremme Translationel Science - UIC Center for klinisk og translationel Sciences Grant UL1TR000050.

1.. Celletransfektion <ol><li> Plate celler dagen før transfektion udpladning færre celler til immunofluorescens. <ol><li> For immunfluorescens: i en 6-brønds plade, frakke dækglas (1 per brønd) med 0,02% gelatine ved 37 ° C i mindst 4 timer, vask en gang med medium og for hver brønd, x 10 5 plade 1 HEK 293T celler i 2 ml komplet vækstmedium [Dulbeccos modificerede Eagles medium (DMEM) plus 10% FBS]. </li><li> Til flowcytometrisk og Western blot-analyser: plade 1,2 x 10 5 HEK 293T celler ...

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	<li>Collura, V., Boissy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+protein-protein+complexes+to+interactomics.">From protein-protein complexes to interactomics.</a> Subcell Biochem. 43, 135-183 (2007).</li><li>Stumpf, 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=Estimating+the+size+of+the+human+interactome.">Estimating the size of the human interactome.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 6959-6964 (2008).</li><li>Shekhawat, S. S., Ghosh, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-protein+systems:+beyond+binary+protein-protein+interactions.">Split-protein systems: beyond binary protein-protein interactions.</a> Curr. Opin. Chem. Biol. 15, 789-797 (2011).</li><li>Dwane, S., Kiely, 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=Tools+used+to+study+how+protein+complexes+are+assembled+in+signaling+cascades.">Tools used to study how protein complexes are assembled in signaling cascades.</a> Bioeng. Bugs. 2, 247-259 (2011).</li><li>Piehler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+methodologies+for+measuring+protein+interactions+in+vivo+and+in+vitro.">New methodologies for measuring protein interactions in vivo and in vitro.</a> Curr. Opin. Struct. Biol. 15, 4-14 (2005).</li><li>Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+bimolecular+fluorescence+complementation+(BiFC)+assays+for+the+visualization+of+protein+interactions+in+living+cells.">Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.</a> Nat. Protoc. 1, 1278-1286 (2006).</li><li>Shyu, Y. J., Suarez, C. D., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+ternary+complexes+in+living+cells+by+using+a+BiFC-based+FRET+assay.">Visualization of ternary complexes in living cells by using a BiFC-based FRET assay.</a> Nat. Protoc. 3, 1693-1702 (2008).</li><li>Wong, K. A., O'Bryan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation.">Bimolecular fluorescence complementation.</a> J. Vis. Exp. (50), e2643 (2011).</li><li>Sugarbaker, E. V., Thornthwaite, J. T., Temple, W. T., Ketcham, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+general+principles+and+applications+to+selected+studies+in+tumor+biology.">Flow cytometry: general principles and applications to selected studies in tumor biology.</a> Int. Adv. Surg. Oncol. 2, 125-153 (1979).</li><li>Koshy, S., Alizadeh, P., Timchenko, L. T., Beeton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+GLUT4+translocation+to+the+plasma+membrane+by+flow+cytometry.">Quantitative measurement of GLUT4 translocation to the plasma membrane by flow cytometry.</a> J. Vis. Exp. (45), e2429 (2010).</li><li>Smith, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+confocal+microscopy.">Basic confocal microscopy.</a> Curr. Protoc. Mol. Biol. Chapter 14 (Unit 14), 11 (2008).</li><li>Westwick, J. K., Lamerdin, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+drug+discovery+with+contextual+assays+and+cellular+systems+analysis.">Improving drug discovery with contextual assays and cellular systems analysis.</a> Methods Mol. Biol. 756, 61-73 (2011).</li><li>Kilpatrick, L. E., Holliday, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+pharmacology+of+G+protein-coupled+receptor+signaling+complexes+using+bimolecular+fluorescence+complementation.">Dissecting the pharmacology of G protein-coupled receptor signaling complexes using bimolecular fluorescence complementation.</a> Methods Mol. Biol. 897, 109-138 (2012).</li></ol>

BiFC er en enkel og følsom metode til at studere protein-protein-interaktion. Denne metode kan ikke anvendes til at identificere nye protein-protein interaktioner, men det er især praktisk at bekræfte protein-protein-interaktion i celler og at studere funktionelle egenskaber, såsom subcellulær lokalisering af protein-komplekser, kortlægning af protein-protein interaktion steder, og til screening af små molekyler / peptider, der kan modulere protein-protein-interaktion. Fordi VN og VC-fragmenter er ikke-fluorescer...

650.000 protein-protein interaktioner skønnes at eksistere i den menneskelige interactome, spiller afgørende roller i at opretholde normal celle-funktioner 1,2. Udover co-immunopræcipitation (co-IP), til guldstandarden studere protein-protein-interaktion fra cellelysat, flere proteinfragment komplementering assays (PCA) er blevet udviklet for at forbedre følsomheden i påvisning protein-protein-interaktion inde celler 3.. Teknikker indbefatter Forster resonans energi overførsel (FRET), bioluminescens resonansenergioverførsel (BRET), og bimolekylær Fluorescens komplementation (BiFC) 4,5. BiFC er baseret på den faciliterede associering af to fragmenter af en fluorescerende protein (her bruger vi Venus, en YFP variant), der hver fusioneret til en potentiel interagerende protein partner (i dette eksempel bruger vi AKAP-Lbc og PDE4D3). Samspillet mellem de to proteiner af interesse i celler resulterer i funktionel fluorescens, som kan visualiseres ved fluorescence mikroskopi med enten levende eller fikserede celler 6,7,8. Sammenlignet med andre PCA&#39;er, BiFC er følsom og mindre teknisk udfordrende, med potentiale til at studere cellulære lokalisering i levende celler. En væsentlig ulempe ved denne teknik er imidlertid, at når der dannes, kan den fluorescerende protein-komplekset ikke vendes. Derfor er det ikke en god metode til at studere dynamiske protein-protein-interaktion. I dette papir, bruger vi BiFC at kortlægge samspillet steder PDE4D3 med AKAP-Lbc ved at overvåge fluorescensintensiteterne af Venus. Sammenlignet med traditionel analyse ved anvendelse af fluorescensmikroskopi, hvilket er tidskrævende og arbejdskrævende (hvis de ikke udføres ved hjælp af automatiserede high throughput maskiner), giver flowcytometri en enkel kvantitativ analyse af tusindvis af celler i en heterogen population over en kort tid 9, 10.. Her, ved at udføre en side-by-side sammenligning af flowcytometrisk-BiFC analyse og traditionelle co-UP, viser vi, at de to metoder levere sammenlignelige data dog flowcytometrisk BiFC analyse ligger mindre tidskrævende og bruger mindre materiale, der kan være mere nyttigt, når celler af interesse er begrænset.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Dulbecco's modified Eagle's media (DMEM)</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin (pen/strep), 100x</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-Flag antibody</td>
			<td>Sigma</td>
			<td>M2, F1804</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-HA antibody</td>
			<td>Covance</td>
			<td>16B12, MMS-101R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&alpha;-tubulin</td>
			<td>Sigma</td>
			<td>DM1A, T9026</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Clontech</td>
			<td>632569</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture plates, 6-well tissue culture treated</td>
			<td>Thermo Fisher Scientific</td>
			<td>130184</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Maxiprep plasmid purification kit, high speed</td>
			<td>Qiagen</td>
			<td>12663</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Phosphate-buffered saline (DPBS), sterile 1x</td>
			<td>Invitrogen</td>
			<td>14190-144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.05% (w/v)</td>
			<td>Gibco</td>
			<td>25300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene round-bottom tubes for FACS staining</td>
			<td>BD Biosciences</td>
			<td>352052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>S74337MF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Prolong gold antifade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P-36931</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superfrost plus Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fisherfinest premium cover glass</td>
			<td>Fisher Scientific</td>
			<td>12-548-5P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>CO2 air-jacketed Incubator</td>
			<td>NuAIR DH autoflow</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Confocal microscope LSM510 META</td>
			<td>Carl Zeiss, Inc</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrophoresis and transfer unit</td>
			<td>Biorad</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cyan ADP Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction

Blandt metoder til at studere protein-protein interaktioner i celler, er bimolekylær Fluorescens Complementation (BiFC) relativt simpel og følsom. BiFC er baseret på produktion af fluorescens under anvendelse af to ikke-fluorescerende fragmenter af et fluorescerende protein (Venus, en gul fluorescerende protein variant, anvendes her). Ikke-fluorescerende Venus fragmenter (VN og VC) fusioneres til to interagerende proteiner (i dette tilfælde AKAP-Lbc og PDE4D3), hvilket giver fluorescens grund VN-AKAP-Lbc-VC-PDE4D3 interaktion og dannelsen af ​​en funktionel fluorescerende protein i celler. BiFC giver oplysninger om den subcellulære lokalisering af protein komplekser og styrken af ​​protein interaktioner baseret på fluorescensintensitet. Men BiFC analyse ved hjælp mikroskopi at kvantificere styrken af ​​protein-protein-interaktion er tidskrævende og noget subjektivt grund heterogenitet i protein-ekspression og interaktion. Ved kobling flowcytometriskanalyse med BiFC metode, kan den fluorescerende BiFC protein-protein-interaktion signal måles nøjagtigt til en stor mængde af celler i en kort tid. Her viser vi en anvendelse af denne metode til at kortlægge områder i PDE4D3 der er nødvendige for interaktion med AKAP-Lbc. Denne high throughput metode kan anvendes til screening faktorer, der regulerer protein-protein-interaktion.

AKAP-Lbc og PDE4D3 konstruktioner (figur 1B) blev fusioneret til VN og VC-fragmenter. Interaktion mellem AKAP-Lbc og PDE4D3 resultater i funktionel YFP fluorescens (figur 1A). Her bruger vi den BiFC metode til at kort AKAP-Lbc-bindingssteder i PDE4D3. Opstrøms konserveret region 1 (UCR1), opstrøms konserveret region 2 (UCR2) og katalytiske region (CAT) er konserveret blandt PDE4 familiens proteiner derfor fuld længde (FL), UCR1 og UCR2 plus CAT blev anvendt til screening af bindingss...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction at JoVE.com

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Flowcytometrisk analyse af bimolekylær Fluorescens komplementation giver en høj produktion kvantitativ metode til at studere protein-protein-interaktion. Denne metode kan anvendes til kortlægning proteinbindingssteder og til screening faktorer, der regulerer protein-protein-interaktion.

Flow cytometri af bimolekylær Fluorescens Complementation: En High Throughput Kvantitativ metode til at studere protein-protein interaktion

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

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