Vorremmo ringraziare Quentina Beatty, Università di Linz, in Austria, per il suo gentile aiuto, Katharina Strub, Università di Ginevra, in Svizzera, per la hCD71-GFP costruire, e Daniel Legler, Università di Costanza, in Svizzera, per il GPI-GFP DAF-costruire. Questo lavoro è stato sostenuto dal Fondo austriaco della scienza (FWF; progetto Y250-B03) e il GEN-AU progetto del Ministero federale austriaco per la Scienza e la ricerca.

Microcontatto stampa: <ol><li> Tagliare campo della micropattern del timbro PDMS con un bisturi </li><li> Lavare il campo contenente il micropattern dal lavaggio con etanolo (100%) e acqua distillata. </li><li> Secco il timbro PDMS con azoto o gas argon. </li><li> Pipettare 50 microlitri BSA-Cy5 soluzione di lavoro (0,67 mg / ml) sul timbro PDMS in modo che l&#39;intero campo micropattern è coperto con una soluzione. Incubare per 30 minuti a temperatura ambiente. </li><li> Lavare il campo micropattern da lavaggio con soluzione tampone fosfato (PBS) e acqua distillata. </li><li> Asciugare il PDMS con azoto o gas argon. </li><li> Inserire il timbro PDMS sotto il proprio peso sul mezzo di una resina epossidica rivestite coprioggetto e incubare per 30 minuti a temperatura ambiente in una capsula di Petri. </li><li> Etichetta la posizione del timbro PDMS sul retro del vetrino e nastri il timbro dalla diapositiva con una pinza. </li><li> Bastone di una scheda Secure camera di ibridazione Sigillo il microcontatto stampato campo sul vetrino. L&#39;etichetta consente di localizzare il centro del campo. </li><li> Pipettare 60 microlitri lavoro soluzione streptavidina (50 mg / ml) nella camera di reazione e incubare il campione per 60 minuti a temperatura ambiente. </li><li> Lavare il campione con 1 ml di PBS con l&#39;aggiunta del tampone in una porta della camera e la rimozione contemporaneamente alla seconda porta con una pompa. </li><li> Pipettare 60 microlitri biotinilato lavoro soluzione di anticorpi (10 mg / ml in PBS integrato con 0,1% di Tween 20; &quot;PBST&quot;) nella camera di reazione e incubare per 60 minuti a temperatura ambiente. </li><li> Lavare il campione con 1 ml PBST e successivamente 1 ml di PBS con l&#39;aggiunta del tampone in una porta della camera e la rimozione di nuovo alla seconda porta con una pompa. </li><li> Conservare le superfici micropatterned in PBS al buio a temperatura ambiente fino a quando le cellule sono pronte per la semina. </li></ol> L&#39;incubazione delle cellule sulla superficie micropatterned: <ol><li> Crescere cellule aderenti al 50% confluenza in un piatto 3 coltura del tessuto cm. </li><li> Staccare cellule che esprimono proteine ​​esca e preda di interesse con l&#39;acido etilendiamminotetraacetico (EDTA), soluzione e centrifugare 5 min a 1.000 giri al minuto. </li><li> Eliminare il supernatante e sciogliere il pellet di cellule nel mezzo di crescita secondo. </li><li> Centrifugare 5 min a 1.000 giri al minuto. </li><li> Eliminare il supernatante e sciogliere il pellet di cellule nel terreno di coltura appropriato. </li><li> Sostituire la PBS dalla camera di reazione sul vetrino micropatterned da 60 ml di sospensione cellulare. </li><li> Creare una camera umidificata immergendo un tampone sterile in acqua distillata e di metterlo in scatole Petri per evitare che il campione di marcia a secco. </li><li> Incubare le cellule durante la notte a 37 ° C in un 5% di CO 2 nell&#39;atmosfera sulle superfici micropatterned. </li><li> Prima di analizzare le cellule al microscopio, cambio del mezzo di soluzione salina tamponata Hank s tra cui Ca 2 + e Mg 2 +. </li></ol> Microscopia: <ol><li> Il coprioggetto è posto su un supporto adatto e la morfologia delle cellule è verificata in luce bianca. </li><li> La qualità del-Cy5 BSA modelli è controllato da eccitazione a 647 nm. </li><li> Microdisegni preda di proteine ​​fluorescenti (GFP o YFP) sono registrati a 488 o 514 nm in riflessione interna totale (TIR) ​​eccitazione. </li></ol> Rappresentante dei risultati: Se c&#39;è interazione tra le proteine ​​bersaglio nelle cellule coltivate su una superficie micropatterned, la preda seguirà la redistribuzione esca. Il micropattern risultante può essere visualizzato dal marchio fluorescenza della proteina preda. È importante sottolineare che il contrasto ottenuto fornisce una misura diretta della forza di interazione (vedi Figura 1). Quindi una semplice valutazione della interazione di due proteine ​​diventa possibile - senza la necessità di elaborare ulteriormente i dati misurati primario. <img src="/files/ftp_upload/1969/1969fig1.jpg" alt="Figura 1" /> Figura 1. Riordino della esche vive nella membrana plasmatica delle cellule a diversi punti di forza di interazione. TIR immagini di cellule transfettate con T24 (a) CD71-GFP su anticorpi CD71-, (b) CD4 e YFP citosolica su CD4-anticorpo e (c) GPI-GFP-DAF sulla CD59-anticorpo microbiochips sono mostrati. I dati sono caratteristici di una forte (a), no (b) o l&#39;interazione debole (c). (A) è riprodotto da (Weghuber et al., In stampa), (b) da (Schwarzenbacher et al., 2008).

<ol>
	<li>Brameshuber, M., Schwarzenbacher, M., Kaltenbrunner, M., Hesch, C., Paster, W., Weghuber, J., Heise, B., Sonnleitner, A., Stockinger, H., Schuetz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reply+to+"Uncoupling+diffusion+and+binding+in+FRAP+experiments.">Reply to "Uncoupling diffusion and binding in FRAP experiments.</a> Nat. Methods. 6, 183-184 (2009).</li><li>Kim, P. W., Sun, Z. J., Blacklow, S. C., Wagner, G., Eck, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Zinc+Clasp+Structure+Tethers+Lck+to+T+Cell+Coreceptors+CD4+and+CD8.">A Zinc Clasp Structure Tethers Lck to T Cell Coreceptors CD4 and CD8.</a> Science. 301, 1725-1728 (2003).</li><li>Ramm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+screening:+a+technology+in+transition.">Image-based screening: a technology in transition.</a> Curr. Opin. Biotechnol. 16, 41-48 (2005).</li><li>Schwarzenbacher, M., Kaltenbrunner, M., Brameshuber, M., Hesch, C., Paster, W., Weghuber, J., Heise, B., Sonnleitner, A., Stockinger, H., Schutz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+for+quantitative+analysis+of+protein-protein+interactions+in+living+cells.">Micropatterning for quantitative analysis of protein-protein interactions in living cells.</a> Nat. Methods. 5, 1053-1060 (2008).</li><li>Weghuber, J., Brameshuber, M., Sunzenauer, S., Lehner, M., Paar, C., Haselbruebler, T., Schwarzenbacher, M., Kaltenbrunner, M., Paster, W., Heise, B., Sonnleitner, A., Stockinger, H., Schutz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+protein-protein+interactions+in+the+live+cell+plasma+membrane+by+quantifying+prey+redistribution+upon+bait+micropatterning.">Detection of protein-protein interactions in the live cell plasma membrane by quantifying prey redistribution upon bait micropatterning.</a> Methods in Enzymology. , .</li></ol>

Il video contiene immagini che sono stati forniti da Olympus, tratto dal sito del Laboratorio di Ricerca di visualizzazione, Brown University o riprodotti da (Kim et al., 2003).

Il video che accompagna illustra un metodo per la rilevazione di interazioni proteina-proteina nel plasma-membrana delle cellule viventi (Schwarzenbacher et al, 2008;. Brameshuber et al, 2009;.. Weghuber et al, in stampa). In linea di principio, qualsiasi TIRF piattaforma basata su microscopia può essere utilizzato come sistema di lettura. Solo quando si vuole un&#39;elevata sensibilità (ad esempio per la rilevazione di singole molecole), microscopi avanzato sarà richiesto. Per ottenere i migliori risultati i seguenti punti critici durante il processo di preparazione richiedono particolare attenzione: <ol class="lroman"><li> Conservare Cy5-stock soluzione in condizioni di buio per evitare lo sbiancamento del fluoroforo e preparare la BSA-Cy5 soluzione lavoro appena prima della stampa microcontact. </li><li> Fare attenzione a non graffiare la micropattern sul PDMS con la punta della pipetta e incubare il campione in condizioni di buio per evitare lo sbiancamento del fluoroforo. </li><li> Mettere il vetrino su un tampone sterile per evitare che si attacchi del vetrino alla capsula di Petri. Una volta che il timbro PDMS ha aderito al vetrino, non muoversi. </li><li> Evitare bolle d&#39;aria nella camera di reazione e incubare il campione in condizioni di buio per evitare lo sbiancamento del fluoroforo. </li><li> Non lasciate che il campione secco per più di 2 minuti per evitare la formazione di cristalli di sale. </li><li> Per il distacco delle cellule non usare tripsina come sarà unirà il dominio extracellulare di proteine ​​di membrana finora espresso. Evitare tripsina sarà utile per le proteine ​​con basso tasso di turnover per formare microdisegni subito dopo l&#39;attaccamento delle cellule sulla superficie. </li><li> Controllare il numero di cellule incubate in un microscopio e fare in modo che le cellule non raggiungono più del 30-50% confluenza. Assicurarsi che le cellule vengono rimosse immediatamente eccessivo a causa del fissaggio rapido delle cellule sulla legate agli anticorpi di superficie. </li><li> Solo con diapositive di alta qualità BSA-Cy5 modelli possono essere utilizzati per ulteriori analisi. </li><li> Espressione stabile della proteina fluorescente preda è preferibile a causa di un maggior numero di cellule che possono essere analizzati per scansione. </li><li> Un&#39;adeguata regolazione TIRF è indispensabile per visualizzare modelli a causa di sfondo citosolico. </li></ol> Il micro-patterning tecnica offre numerose opportunità per l&#39;analisi delle interazioni proteina-proteina. In primo luogo, insieme con la quantificazione dei locali, risolta spazialmente interazioni proteina-proteina anche la rilevazione delle interazioni deboli o indiretta è possibile senza lo svantaggio di dare un elevato numero di falsi positivi o negativi. In secondo luogo consente al ricercatore di analizzare le interazioni proteina-proteina nel plasma-membrana di cellule vive, che è difficile da raggiungere da approcci biochimici come il 2-ibrido schermo. In terzo luogo l&#39;approccio consente la rilevazione di esca-preda interazioni che sono modulati da cambiamenti ambientali come la temperatura, la presenza di diverse proteine ​​o altre molecole o modificazioni post-traduzionali. Così il test permette di modulatori proiezione di una coppia data interazione nel contesto delle cellule vive. Inoltre, riducendo la densità superficiale del ligando cattura o l&#39;uso di leganti monovalente, l&#39;analisi dello stato di riposo diventa possibile. Infine, se adeguatamente le piattaforme di scansione vengono utilizzati, il numero di cellule analizzate è abbastanza alto per soddisfare domande throughput elevato delle aziende farmaceutiche per lo screening di stupefacenti (Ramm, 2005).

in-vivo detection of protein-protein interactions on micro-patterned surfaces

Svelare la rete di interazione delle molecole<em&gt; In-vivo</em&gt; È la chiave per la comprensione dei meccanismi che regolano la funzione delle cellule e del metabolismo. Una moltitudine di opzioni metodologiche per affrontare le interazioni molecolari nelle cellule sono state sviluppate, ma la maggior parte di questi metodi soffrono di essere un po &#39;indiretto, e quindi difficilmente quantitativo. Al contrario, alcuni approcci quantitativi di fascia alta sono stati introdotti, che però sono difficili da estendere ad elevato throughput. Per combinare le capacità di throughput elevato con la possibilità di estrarre informazioni quantitative, abbiamo recentemente sviluppato un nuovo concetto per identificare interazioni proteina-proteina (Schwarzenbacher<em&gt; Et al</em&gt;., 2008). Qui, descriviamo un protocollo dettagliato per la progettazione e la costruzione di questo sistema che permette di analizzare le interazioni tra un fluoroforo marcata con proteine ​​(&quot;preda&quot;) e una proteina di membrana (&quot;esca&quot;)<em&gt; In-vivo</em&gt;. Le cellule sono placcati su superfici micropatterned funzionalizzati con anticorpi contro il dominio esca exoplasmic. Bait-preda interazioni sono stati analizzati attraverso la redistribuzione della preda fluorescenti. Il metodo è caratterizzato da elevata sensibilità fino al livello di singole molecole, la capacità di rilevare le interazioni deboli, e la capacità di throughput elevato, il che rende applicabile come strumento di screening.

Watch this Scientific Journal Video about In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces at JoVE.com

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

Questo video mostra esperimenti con successiva analisi delle interazioni proteina-proteina mediante l&#39;uso di micro-fantasia superfici. L&#39;approccio offre la possibilità di rilevare le interazioni delle proteine ​​nelle cellule viventi e combina le elevate capacità di throughput con la possibilità di estrarre informazioni quantitative.

In vivo di rilevamento di interazioni proteina-proteina su disegno micro, Superfici

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

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Research

JoVE Journal

Biology

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

10.7K Views.  Johannes Kepler Universitat Linz. Questo video mostra esperimenti con successiva analisi delle interazioni proteina-proteina mediante l'uso di micro-fantasia superfici. L'approccio offre la possibilità di rilevare le interazioni delle proteine ​​nelle cellule viventi e combina le elevate capacità di throughput con la possibilità di estrarre informazioni quantitative.

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

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-Ubiquitina membrana a base di lievito Due-Hybrid (MITO) Sistema: un potente strumento per identificare interazioni proteina-proteina

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Ricerca

Biologia

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Imaging interazioni proteina-proteina In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

Quantificazione in vivo di G interazioni proteina recettore accoppiato con spettralmente risolte microscopia a due fotoni

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

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.

Un protocollo per l&#39;identificazione di interazioni proteina-proteina Sulla base di<sup&gt; 15</sup&gt; N marcatura metabolica, immunoprecipitazione, Spettrometria di massa quantitativa e di affinità di modulazione

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

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.

Interazioni proteina-proteina visualizzati tramite bimolecular complementazione fluorescenza in protoplasti di tabacco e foglie

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.

Indagare interazioni proteina-proteina in cellule vive utilizzando il trasferimento bioluminescenza Resonance Energy

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

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

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

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

Probing ad alta densità di microarrays della proteina funzionali per rilevare le interazioni proteina-proteina

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

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

Sviluppo di inibitori di interazioni proteina-proteina attraverso REPLACE: Applicazione alla progettazione e allo sviluppo non ATP inibitori CDK competitivo

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Immunoprecipitazione della cromatina Saggio per la identificazione di Arabidopsis interazioni proteina-DNA<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Studio delle interazioni proteina-proteina nella ricerca di autofagia

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...