Wir bedanken uns bei Vi Nguyen für die Durchsicht des Manuskripts und allgemeine Labor Unterstützung.

1. Vorbereitung von Agrobacterium Kultur <ol><li> Agrobacterium-Stamm GV3101 zuvor mit Gateway kompatibel BIFC Vektor pEarleyGate201-YC und pEarleyGate202-YN, die jeweils eine Verschmelzung von GOI Konstrukt transformiert. </li><li> Impfen einer 5-ml YEB (5g L -1 Beef-Extrakt, 1g L -1 Hefeextrakt, 5 g L -1 Pepton, 5 g L -1 Saccharose, 2 mM MgSO 4, pH 7,2) Kultur BIFC Fusionsprotein-Konstrukten transformiert Agrobacterium mit dem einer angemessenen Antibiotika-Auswahl. Wachsen Kultur über Nacht bei 28 ° C unter Schütteln bei 200 Umdrehungen pro Minute. </li><li> Nehmen Sie 1 ml der Kultur in ein steriles 1,5 ml Zentrifugenröhrchen. </li><li> Pellet-Zellen bei 1.000 g für 10 min bei Raumtemperatur, Überstand verwerfen. </li><li> Waschen des Pellets durch Zugabe von 1 ml der Infiltration Medien (5 g L -1 D-Glucose, 50 mM MES (Sigma), 2 mM Na 3 PO 4, 0,1 mM Acetosyringon) 11. Pellet durch Pipettieren, Pellet-Zellen wieder bei 1.000 g für 10 min. </li><li> Wiederholen Sie den Waschvorgang zwei weitere Male. </li><li> Das Pellet in 0,5 ml Infiltration Medien. </li><li> Die Absorption bei 600 nm und passen endgültigen OD 600 bis 1,0 bis 1,2. </li><li> Aliquot einer gleichen Menge von Agrobacterium Suspension von jedem Konstrukt in ein neues 1,5-ml-Tube, Wirbel für 10 sek. Für eine einzelne Infiltration, ist 100 ul von jedem Konstrukt mehr als genug. </li><li> Die Agrobacterium-Gemisch ist nun bereit für die Infiltration. </li></ol> 2. Infiltration <ol start="11"><li> N. benthamiana Pflanzen werden bei 21 ° C gewachsen, mit 16-h Licht und 8-h Dunkel-Zyklen. Fünf bis sechs Wochen alten Pflanzen sollten für die Infiltration verwendet werden. </li><li> Entfernen Sie Pflanzen aus dem Wachstum Raum und unter weißem Licht für 1 h vor infiltratring so dass die Spaltöffnungen vollständig zu öffnen. </li><li> Erstellen Sie resuspendiert Agrobakterium Mischung in eine 1-ml Slip-Spitze Tuberkulinspritze ohne Nadel auf. </li><li> Setzen Sie die Spitze der Spritze an die Unterseite des Blattes und sanft drücken Sie den Kolben, während die direkte Unterstützung der Oberseite des Blattes mit einem Finger. Die Flüssigkeit wird durch das Blatt diffuse, wie es die mesophyllar Lufträume füllt. </li><li> Beschriften Sie die infiltrierten Bereich mit einem Filzstift für die künftige Identifikation. </li><li> Wenn Infiltration mit unterschiedlichen Konstrukten, vergewissern Sie sich, um entweder Ihre Handschuhe oder reinigen Sie sie mit 70% Ethanol zwischen Infiltrationen. Wenn möglich, lassen Sie eine Mittelrippe zwischen verschiedenen Proben, um eine Kreuzkontamination zu vermeiden. </li><li> Legen Sie Pflanzen in Wachstum Kabinett unter normalen Wachstumsbedingungen. </li></ol> 3. Die Beobachtung des rekonstituierten YFP-Signal <ol start="18"><li> Excise einem 5mm x 5mm Segment Blattgewebe innerhalb der infiltrierten Zone </li><li> Montieren Sie die Probe im Wasser, auf einem Glasobjektträger, decken Sie die Probe mit einem Deckglas und untersuchen die Wechselwirkungen mit einem konfokalen Fluoreszenzmikroskop oder. </li><li> Expression sollte alle 24 Stunden ab dem Zeitpunkt der Infiltration bis zu 5 Tage später beobachtet werden, wie Überexpression / Handel mit fluoreszierenden Fusionsproteinen Anzeige verschiedenen Orten über einen zeitlichen Verlauf führen kann. </li></ol> 4. Repräsentative Ergebnisse: Ein Beispiel für Protein-Protein-Interaktion durch BIFC Assay getestet wird in Abbildung 1 dargestellt. AtTHP1 und AtSAC3A werden geglaubt, um Komponenten der Arabidopsis-Homolog der Hefe TREX-2-Komplex. Sie können mit einem nuclearporin AtNUP1 interagieren und zu erleichtern mRNA Export 10. AtTHP1 und AtSAC3A wurden in pEarlyGate201-YC geklont während AtNUP1 in pEarlyGate202-YN geklont wurde. Die Konstrukte wurden anschließend in GV3101 transformiert. (; AtTHP1 + AtSAC3A; AtNUP1 + AtSAC3A AtTHP1 + AtNUP1) für die Infiltration der drei Konstrukte wurden mit 3 möglichen Kombinationen gepaart. Die rekonstituierte YFP-Signal wurde unter einem LCSM 48 Stunden nach der Infiltration beobachtet. Die Signale wurden in den epidermalen Zellen beobachtet und lokalisiert in der nuklearen Peripherie oder nuklearen Plasma, das im Einklang mit dem sub-zelluläre Lokalisation dieser Proteine ​​ist. Die negativen Kontrollen zeigten keine YFP-Signal. <img src="/files/ftp_upload/3473/3473fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Arabidopsis TREX-2-mRNA export komplexen Komponenten miteinander interagieren. N. benthamiana Blätter, mit Konstrukten des GOI cotransformiert fusioniert pEarleyGate201-YC und pEarleyGate202-YN (wie angegeben), wurden abgebildet 48-72 h nach der Infiltration mit einem Leica TCS SP2 confocol Mikroskop. Die Bilder werden als fusionierte konfokalen YFP und Hellfeld-Bilder von epidermalen N. gezeigt benthamiana Blattzellen

<ol>
	<li>Ohad, N., Yalovsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+bimolecular+fluorescence+complementation+(BiFC)+to+assay+protein-protein+interaction+in+plants.">Utilizing bimolecular fluorescence complementation (BiFC) to assay protein-protein interaction in plants.</a> Methods in molecular biology. 655, 347-358 (2010).</li><li>Fang, Y., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BiFC+imaging+assay+for+plant+protein-protein+interactions.">BiFC imaging assay for plant protein-protein interactions.</a> Cold Spring Harbor protocols. 2, (2010).</li><li>Schutze, K., Harter, K., Chaban, C. <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+(BiFC)+to+study+protein-protein+interactions+in+living+plant+cells.">Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in living plant cells.</a> Methods in molecular biology. 479, 189-202 (2009).</li><li>Hiatt, S. M. <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+(BiFC)+analysis+of+protein+interactions+in+Caenorhabditis+elegans.">Bimolecular fluorescence complementation (BiFC) analysis of protein interactions in Caenorhabditis elegans.</a> Methods. 45, 185-191 (2008).</li><li>Barnard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+implementation+of+split-GFP-based+bimolecular+fluorescence+complementation+(BiFC)+assays+in+yeast.">Development and implementation of split-GFP-based bimolecular fluorescence complementation (BiFC) assays in yeast.</a> Biochemical Society Transactions. 36, 479-482 (2008).</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> Nature. 1, 1278-1286 (2006).</li><li>Hu, C. D., Grinberg, A. V., 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=Visualization+of+protein+interactions+in+living+cells+using+bimolecular+fluorescence+complementation+(BiFC)+analysis.">Visualization of protein interactions in living cells using bimolecular fluorescence complementation (BiFC) analysis.</a> Current protocols in cell biology. 21, 3-3 (2006).</li><li>Earley, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gateway-compatible+vectors+for+plant+functional+genomics+and+proteomics.">Gateway-compatible vectors for plant functional genomics and proteomics.</a> The Plant journal : for cell and molecular biology. 45, 616-629 (2006).</li><li>Karimi, M., Inze, D., Depicker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATEWAY+vectors+for+Agrobacterium-mediated+plant+transformation.">GATEWAY vectors for Agrobacterium-mediated plant transformation.</a> Trends in Plant Science. 7, 193-195 (2002).</li><li>Lu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+homolog+of+the+yeast+TREX-2+mRNA+export+complex:+components+and+anchoring+nucleoporin.">Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin.</a> The Plant journal : for cell and molecular biology. 61, 259-270 (2010).</li><li>Sparkes, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=transient+expression+of+fluorescent+fusion+proteins+in+tobacco+plants+and+generation+of+stably+transformed+plants.">transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants.</a> Nature protocols. 1, 2019-2025 (2006).</li><li>Rackham, O., Brown, C. M. <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+RNA-protein+interactions+in+living+cells:+FMRP+and+IMP1+interact+on+mRNAs.">Visualization of RNA-protein interactions in living cells: FMRP and IMP1 interact on mRNAs.</a> The EMBO journal. 23, 3346-3355 (2004).</li><li>Demidov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+complementation+of+split+fluorescent+protein+triggered+by+DNA+hybridization.">Fast complementation of split fluorescent protein triggered by DNA hybridization.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 2052-2056 (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> Nature. 3, 1693-1702 (2008).</li></ol>

Keine Interessenskonflikte erklärt.

BIFC Assay ist ein leistungsfähiges Werkzeug für das Studium Protein-Interaktionen. Im Gegensatz zu den traditionellen Y2H Assay BIFC ermöglicht nicht nur die Visualisierung von Protein-Protein-Interaktionen, sondern bietet auch weitere Informationen des Protein-Komplexes, wie sub-zelluläre Lokalisation. Auch ist es möglich, indirekten Wechselwirkungen so lange zu erkennen, wie die beiden Kandidaten Proteine ​​nahe genug von einem dritten Partner gebracht werden. Wie jede Technologie hat BIFC seine Grenzen. Auf...

<table border='1'>
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Sigma</td>
			<td>D134406</td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma</td>
			<td>M2933</td>
		</tr>
		<tr>
			<td>1 mL slip-tip tuberculin syringe</td>
			<td>BD</td>
			<td>309602</td>
		</tr>
	</tbody>
</table>

detection of protein interactions in plant using a gateway compatible bimolecular fluorescence complementation (bifc) system

Wir haben eine BIFC Technik, um die Interaktion zwischen zwei Proteinen in vivo-Test entwickelt. Dies wird durch Aufteilung eines gelb fluoreszierenden Proteins (YFP) in zwei nicht-überlappenden Fragmenten durchgeführt. Jedes Fragment wird in-frame, ein Gen von Interesse geklont. Diese Konstrukte können dann in Nicotiana benthamiana werden via Agrobacterium vermittelte Transformation co-transformiert, die den Transit Expression von Fusionsproteinen. Die Rekonstitution von YFP-Signal erfolgt nur, wenn der gerichtlichen Untersuchung Proteine ​​1-7 interagieren. Um zu testen und zu validieren das Protein-Protein-Interaktionen können BIFC zusammen mit Hefe-Zwei-Hybrid (Y2H)-Assay verwendet werden. Dies kann erkennen indirekten Wechselwirkungen, die in der Y2H übersehen werden kann. Gateway-Technologie ist eine universelle Plattform, die Forschern die Shuttle-Gens von Interesse (GOI) in so viele Expression und funktionelle Analyse-Systeme wie möglich zu 8,9. Sowohl die Orientierung und Leserahmen können ohne Verwendung von Restriktionsenzymen oder Ligation zum Ausdruck bereit-Klone machen aufrechterhalten werden. Als Ergebnis kann man beseitigen alle Re-Sequenzierung Schritte, um konsistente Ergebnisse in den Experimenten zu gewährleisten. Wir haben eine Reihe von Gateway kompatibel BIFC und Y2H Vektoren, die Forscher stellen mit einfach zu bedienenden Tools zu führen sowohl BIFC und Y2H Assays 10 erstellt. Hier zeigen wir die einfache Nutzung unserer BIFC System zum Protein-Protein-Interaktionen in N. Test benthamiana Pflanzen.

Watch this Scientific Journal Video about Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System at JoVE.com

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

Wir haben eine Technik, um Protein-Protein-Interaktionen in Anlage-Test entwickelt. Ein gelb fluoreszierendes Protein (YFP) ist in zwei nicht überlappende Fragmente aufgeteilt. Jedes Fragment wird in-frame, ein Gen von Interesse via Gateway-System geklont, wodurch Expression von Fusionsproteinen. Rekonstitution der YFP-Signal erfolgt nur, wenn der gerichtlichen Untersuchung Proteinen interagieren.

Detektion von Protein-Wechselwirkungen in Plant mit einem Gateway kompatibel bimolekularen Fluoreszenz Komplementation (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 ...

detection-protein-interactions-plant-using-gateway-compatible

Research

JoVE Journal

Biology

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

25.2K Aufrufe.  University of Western Ontario. Wir haben eine Technik, um Protein-Protein-Interaktionen in Anlage-Test entwickelt. Ein gelb fluoreszierendes Protein (YFP) ist in zwei nicht überlappende Fragmente aufgeteilt. Jedes Fragment wird in-frame, ein Gen von Interesse via Gateway-System geklont, wodurch Expression von Fusionsproteinen. Rekonstitution der YFP-Signal erfolgt nur, wenn der gerichtlichen Untersuchung Proteinen interagieren.

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

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

Schnelle Homogene Detektion von biologischen Assays mit magnetischen Modulation Biosensorik-System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Forschung

Biologie

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Bimolekularen Fluoreszenz Komplementation (BIFC) Assay für Protein-Protein-Wechselwirkungen in Onion Zellen unter Verwendung des Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

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.

In-vivo-Detektion von Protein-Protein-Interaktionen auf Micro-gemusterten Oberflächen

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

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.

Bimolekularen Fluoreszenz Komplementation

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

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Ein Protokoll für die Identifizierung von Protein-Protein-Wechselwirkungen Basierend auf<sup&gt; 15</sup&gt; N Metabolische Markierung, Immunpräzipitation, Quantitative Massenspektrometrie und Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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.

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.

Durchflusszytometrieanalyse bimolekularer Fluorescence Komplementierung: A High Throughput Quantitative Methode zur Protein-Protein Interaction Study

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

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.

Protein-Protein-Wechselwirkungen durch Bimolekulare Fluoreszenz-Komplementation in Tabak-Protoplasten-und Blätter-Visualisierte

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

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Untersuchung von Protein-Protein-Interaktionen in lebenden Zellen Mit Biolumineszenz-Resonanz-Energie-Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

Bestimmung der Protein-Ligand-Wechselwirkungen mit Differential Scanning Fluorimetrie

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

Bestimmen Membrane Protein Topologie Mit Fluorescence Protease Schutz (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...