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 cells/well in 2 ml complete growth medium in 6-well plate (0.3 x 105 HEK 293T cells in 0.5 ml complete growth medium per well in 24-well plate).</li>
	</ol>
	</li>
	<li>Prepare DNA for transfection according to manufacture's protocol: Effectene (Qiagen) is used for immunofluorescence. TransIT-LT1 (Mirus) is used for flow cytometric and Western blot analyses. Amount of DNA used is listed below.
	<table border="1">
		<tbody>
			<tr>
				<td>&nbsp;</td>
				<td>24-well plate (ng)</td>
				<td>6-well plate (ng)</td>
			</tr>
			<tr>
				<td>CFP-vector</td>
				<td>10</td>
				<td>50</td>
			</tr>
			<tr>
				<td>VNN-AKAP-Lbc</td>
				<td>200</td>
				<td>1000</td>
			</tr>
			<tr>
				<td>VCN-PDE4D3-FL(full-length PDE4D3)</td>
				<td>2.5, 5, 10, 20, 40, 80</td>
				<td>100</td>
			</tr>
			<tr>
				<td>VCN-PDE4D3-UCR1(a PDE4D3 N-terminal fragment containing the UCR1 domain)</td>
				<td>&nbsp;</td>
				<td>100</td>
			</tr>
			<tr>
				<td>VCN-PDE4D3-UCR2+CAT (a PDE4D3 C-terminal fragment containing the UCR2 and catalytic domains)</td>
				<td>&nbsp;</td>
				<td>100</td>
			</tr>
		</tbody>
	</table>
	Transfection using Effectene (6-well):
	<ol>
		<li>Add indicated amount of plasmid DNA + 4 &mu;l enhancer to 100 &mu;l buffer EC, mix, and incubate for 5 min at room temperature.</li>
	 <li>Add 5 &mu;l Effectene, mix, and incubate for 10 min at room temperature. Add drop-wise to cells.	 </li>
 </ol>
 Transfection using TransIT-LT1 (6-well/24-well):
 <ol>
		<li>Add indicated amount of plasmid DNA to 250 &mu;l/50 &mu;l of Opti-MEM I serum-free medium in a sterile tube.</li>
		<li>Add TransIT-LT1 reagent to the diluted DNA mixture and pipette gently to mix. (TransIT-LT1 Reagent:DNA ratio is 3 &mu;l of TransIT-LT1 reagent per 1 &mu;g of DNA).</li>
		<li>Incubate for 30 min at room temperature and add drop-wise to cells.</li>
	</ol>
 </li>
 </ol>
2. Cell Preparation for Flow Cytometry, Western Blot, and Immunofluorescence
<ol>
	<li>24 hr post transfection, check fluorescence under epifluorescence microscope before harvesting cells.</li>
	<li>Cell preparation for flow cytometric analysis:
	<ol>
		<li>Wash cells with 0.5 ml/2 ml ice-cold PBS for 24-well/6-well plates.</li>
		<li>Detach cells using 50 &mu;l/250 &mu;l 0.05% trypsin.</li>
		<li>Neutralize trypsin with 200 &mu;l/1 ml DMEM medium.</li>
		<li>Collect 250 &mu;l cells (for both 24-well and 6-well plate) by centrifugation at 500 x g for 5 min, use the remaining cells (1 ml) for Western blot analysis.</li>
		<li>Resuspend cells in 250 &mu;l FACS buffer [PBS + 0.5% (w/v) BSA + 2 mM EDTA], store on ice for flow cytometric analysis. Cells can also be fixed in 1% paraformaldehyde (in PBS) if flow cytometry analysis will not be immediate.</li>
	</ol>
	</li>
	<li>Cell preparation for Western blot analysis: carried out in parallel with cell preparation for flow cytometry analysis.
	<ol>
		<li>Wash cells 1x with 2 ml ice-cold PBS, lyse cells by adding 100 &mu;l of cell lysis buffer [10 mM sodium phosphate buffer, pH 6.95, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100].</li>
		<li>Collect cell lysate by centrifugation for 10 min at 21,000 x g at 4 &deg;C.</li>
		<li>Quantify protein concentration by Bradford protein assay, load equal amounts of protein per lane for SDS-PAGE, and transfer to nitrocellulose for immunoblotting.</li>
	</ol>
	</li>
	<li>Cell preparation for immunofluorescence.
	<ol>
		<li>Rinse cells 3x with 2 ml PBS, fix cells in 1 ml of 3.7% paraformaldehyde (in PBS) for 10 min at room temperature, then wash 3x with 2 ml PBS for 5 min.</li>
		<li>Mount cover slip on a glass slide, using mounting medium. Seal the coverslip edges using nail polish if necessary.</li>
		<li>Store the slides at 4 &deg;C prior to examination.</li>
	</ol>
	</li>
</ol>
3. Flow Cytometry and Immunofluorescence Microscopy
<ol>
	<li>Flow cytometry is performed using a Beckman Coulter Cyan ADP, equipped with 488 nm, 405 nm, and 642 nm solid-state lasers. Summit software is used for acquisition and analysis.
	<ol>
		<li>Calibrate flow cytometer using standard beads.</li>
		<li>Plot Forward scatter (FSC)/Sideward scatter (SSC) on a linear scale for gating live cell population.</li>
		<li>Plot FSC/voltage pulse width for gating non-aggregated live cell population.</li>
		<li>Plot count/FL6 (CFP fluorescence, 405 nm) on a log scale for gating non-aggregated live CFP-positive cells.</li>
		<li>Plot count/FL1 (YFP fluorescence, 488 nm) on a log scale for BiFC intensity.</li>
		<li>Run YFP-CFP- double negative samples, adjust FSC, SSC, FL1 and FL6 photo multiplier tube (PMT) voltage to set the threshold for each signal.</li>
		<li>Run single positive (both YFP+ and CFP+) samples for future off-line compensation</li>
		<li>Acquire at least 10,000 gated events (non-aggregated live CFP+ cells) for analysis.</li>
	</ol>
	</li>
	<li>Immunofluorescence microscopy is performed using a Zeiss LSM 510 confocal microscope. Note: it is important to keep all settings (such as the zoom, pinhole, detector gain, amplifier offset, frame size, scan speed, scan average, and laser power) consistent between samples for proper comparison of all data.</li>
</ol>
4. Data Analysis (Performed Using Summit Software)
<ol>
	<li>Set up the analysis protocol similar to that for acquisition with proper gating: 
	Gate 1 in FSC/SSC dot plot for live cells. 
	Gate 2 in FSC/pulse width of Gate 1 for non-aggregated live cells. 
	Gate 3 in count/CFP of Gate 3 for non-aggregated CFP+ live cells.</li>
	<li>Compensate YFP and CFP signal using YFP+ and CFP+ single positive cells.</li>
	<li>Re-determine cut-off lines for CFP/YFP-positive cells.</li>
	<li>Export YFP mean fluorescence intensity (MFI) of CFP+ cells to Excel for data-plotting.</li>
	<li>Normalize YFP MFI to expression levels of AKAP-Lbc, PDE4D3, and loading control &alpha;-tubulin, as determined by Western blot.</li>
</ol>

<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>

The authors declare that they have no competing financial interests.

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><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>Dulbecco&amp;#8217;s modified Eagle&amp;#8217;s media (DMEM)</td><td>Invitrogen</td><td>11965-092</td><td></td></tr><tr><td>Penicilin-Streptomycin (pen/strep), 100x</td><td>Invitrogen</td><td>15070-063</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Invitrogen</td><td>16000-044</td><td></td></tr><tr><td>Anti-Flag antibody</td><td>Sigma</td><td>M2, F1804</td><td></td></tr><tr><td>Anti-HA antibody</td><td>Covance</td><td>16B12, MMS-101R</td><td></td></tr><tr><td>&amp;#945;-tubulin</td><td>Sigma</td><td>DM1A, T9026</td><td></td></tr><tr><td>Anti-GFP antibody</td><td>Clontech</td><td>632569</td><td></td></tr><tr><td>Cell culture plates, 6-well tissue culture treated</td><td>Thermo Fisher Scientific</td><td>130184</td><td></td></tr><tr><td>HEK 293T cells</td><td>ATCC</td><td>CRL-11268</td><td></td></tr><tr><td>Maxiprep plasmid purification kit, high speed</td><td>Qiagen</td><td>12663</td><td></td></tr><tr><td>Dulbecco&amp;#8217;s Phosphate-buffered saline (DPBS), sterile 1x</td><td>Invitrogen</td><td>14190-144</td><td></td></tr><tr><td>Trypsin-EDTA, 0.05% (w/v)</td><td>Gibco</td><td>25300</td><td></td></tr><tr><td>Polystyrene round-bottom tubes for FACS staining</td><td>BD Biosciences</td><td>352052</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>S74337MF</td><td></td></tr><tr><td>Prolong gold antifade reagent with DAPI</td><td>Invitrogen</td><td>P-36931</td><td></td></tr><tr><td>Superfrost plus Microscope slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Fisherfinest premium cover glass</td><td>Fisher Scientific</td><td>12-548-5P</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; air-jacketed Incubator</td><td>NuAIR DH autoflow</td><td></td><td></td></tr><tr><td>Confocal microscope LSM510 META</td><td>Carl Zeiss, Inc</td><td></td><td></td></tr><tr><td>Electrophoresis and transfer unit</td><td>Biorad</td><td></td><td></td></tr><tr><td>Cyan ADP Flow Cytometer</td><td>Beckman Coulter</td><td></td><td></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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Research

JoVE Journal

Biology

18.3K Views.  University of Illinois at Chicago. 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.

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

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Biochemistry

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

Bioengineering

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

Developmental Biology

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Immunology and Infection

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

FLIM-FRET Imaging for Characterization of Protein-Protein Interactions in Live Bacteria

Source: Manko, H. et al., <a href="https://app.jove.com/v/61602">FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.</a> J. Vis. Exp. (2020)
The video describes the FLIM-FRET imaging technique to determine the protein-protein interaction in live bacteria expressing cytoplasmic proteins labeled with fluorescent proteins, a donor eGFP, and acceptor mCherry. The combined technique also allows the quantification of the interacting proteins.

Source: Manko, H. et al., FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria. J. Vis. Exp. (2020)
The video describes the FLIM-FRET ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-protein interactions

Bimolecular Fluorescence Complementation-Coupled Photoactivated Localization Microscopy

Source: Nickerson, A. et al., <a href="https://app.jove.com/v/53154">Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM).</a> J. Vis. Exp. (2015)
This video describes BiFC-PALM &#8213; a combination of photoactivated localization microscopy and bimolecular fluorescence complementation &#8213; to assess protein-protein interactions. BiFC involves the fusion of two fluorescent protein fragments with two interacting proteins of interest. When the two proteins interact, the fragments are brought into proximity, which allows the two parts to come together and reconstitute a functional fluorescent protein. The fluorescence of a single fluorophore is localized at a single molecule level inside cells by PALM.

Source: Nickerson, A. et al., Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM). J. Vis. Exp. (2015)
This ...

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

Summary

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Chapters in this video

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Bimolecular Fluorescence Complementation

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

FLIM-FRET Imaging for Characterization of Protein-Protein Interactions in Live Bacteria

Bimolecular Fluorescence Complementation-Coupled Photoactivated Localization Microscopy