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

We thank the O'Bryan lab at UIC for critical experimental evaluation and discussion. This work was supported by American Heart Association Grant 11SDG5230003 to GKC and National Center for Advancing Translational Science - UIC Center for Clinical and Translational Sciences Grant UL1TR000050.

1. Cell Transfection
<ol>
	<li>Plate cells the day before transfection, plating fewer cells for immunofluorescence.
	<ol>
		<li>For immunofluorescence: in a 6-well plate, coat glass cover slips (1 per well) with 0.02% gelatin at 37 &deg;C for at least 4 hr, wash once with medium, and for each well, plate 1 x 105 HEK 293T cells in 2 ml complete growth medium [Dulbecco's modified Eagle's media (DMEM) plus 10% FBS].</li>
		<li>For flow cytometric and Western blot analyses: plate 1.2 x 105 HEK 293T ce...

<ol>
	<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 is a simple and sensitive method to study protein-protein interaction. This method cannot be used to identify new protein-protein interactions, however, it is especially convenient to confirm protein-protein interaction inside cells and to study functional properties; such as subcellular localization of protein complexes, mapping of protein-protein interaction sites, and for screening of small molecules/peptides that can modulate protein-protein interaction. Because VN and VC fragments are non-fluorescent, backgroun...

650,000 protein-protein interactions are estimated to exist in the human interactome, playing critical roles in maintaining normal cell functions 1,2. Besides co-immunoprecipitation (co-IP), the gold standard to study protein-protein interaction from cell lysate, several protein fragment complementation assays (PCA) have been developed to improve sensitivity in detecting protein-protein interaction inside cells 3. Techniques include F&ouml;rster resonance energy transfer (FRET), Bioluminescence Resonance Energy Transfer (BRET), and Bimolecular Fluorescence Complementation (BiFC) 4,5. BiFC is based on the facilitated association of two fragments of a fluorescent protein (here we use Venus; a YFP variant) that are each fused to a potential interacting protein partner (in this example we use AKAP-Lbc and PDE4D3). Interaction of the two proteins of interest inside cells results in functional fluorescence, which can be visualized by fluorescence microscopy using either live or fixed cells 6,7,8. Compared to other PCAs, BiFC is sensitive and less technically challenging, with potential to study the cellular localization in live cells. A major drawback of this technique however is that once formed, the fluorescent protein complex cannot be reversed. Therefore it is not a good method to study dynamic protein-protein interaction. In this paper, we use BiFC to map the interaction sites of PDE4D3 with AKAP-Lbc by monitoring the fluorescence intensities of Venus. Compared to traditional analysis using fluorescence microscopy, which is time-consuming and labor-intensive (if not carried out using automated high throughput machinery), flow cytometry provides a straightforward quantitative analysis of thousands of cells in a heterogeneous population over a short time 9,10. Here, by carrying out a side-by-side comparison of flow cytometric-BiFC analysis and traditional co-IP, we demonstrate that the two methods provide comparable data, however, flow cytometric BiFC analysis is less time consuming and uses less material which may be more useful when cells of interest are limited.

<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

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

AKAP-Lbc and PDE4D3 constructs (Figure 1B) were fused to VN and VC fragments, respectively. Interaction of AKAP-Lbc and PDE4D3 results in functional YFP fluorescence (Figure 1A). Here we use the BiFC method to map AKAP-Lbc-binding sites in PDE4D3. Upstream conserved region 1 (UCR1), upstream conserved region 2 (UCR2) and catalytic region (CAT) are conserved among PDE4 family proteins, therefore, full length (FL), UCR1 and UCR2 plus CAT were used for screening the binding site of PDE4D3 t...

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

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

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

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