Ringraziamo Drs. Steve Monte e Xiaoning Zhang per il 35S:: GFP pGlowbug costruire e la Fellowship Hokensen dottorato di ricerca in laboratorio CH Z. L&#39;s è supportato dal US National Science Foundation (IOB0616096 e MCB0744752). ZL è parzialmente supportata dalla University of Maryland Stazione esperimento agricola.

I. Preparazione plasmidi Il DNA plasmide usato per bombardamenti devono essere di purezza elevata e ad una concentrazione di 500ng/μl o superiore. Per BiFC che coinvolgono due costrutti plasmide, 25 mcg di ogni plasmide è necessario. In altre parole, 1:1 rapporto molare dei due plasmidi deve essere miscelato per dare origine a 50 mg di DNA totale plasmide per la preparazione di cartucce. Siate consapevoli che l&#39;aggiunta di più del DNA di 50 mg può causare agglomerazione delle particelle d&#39;oro e deve essere evitato. Disponibili in commercio i kit di estrazione del DNA plasmidico sono raccomandati per isolare DNA plasmide. Vettori plasmidici dovrebbe essere relativamente di piccole dimensioni come pUC19. Abbiamo clonato SEU e LUH cDNA in pUC19-SPYNE e pUC19-SPYCE, rispettivamente 3. Vettori binari per la trasformazione mediata da Agrobacterium sono troppo grandi e inadeguate per il bombardamento. II. Cartuccia preparazione Preparazione cartuccia coinvolge particelle di rivestimento d&#39;oro con il DNA plasmidico e di caricarli in tubi di plastica che viene successivamente tagliato in pezzi mezzo pollice di lunghezza, che può essere caricato nel titolari gene pistola cartuccia. Preparazione della cartuccia è descritto in dettaglio grande in un articolo separato Giove 4 e, quindi, non descritto qui. Per il bombardamento di cellule di cipolla, si consiglia di mescolare 50 mg di DNA plasmidico con 12,5 mg di particelle d&#39;oro per la preparazione, che produce circa un massimo di 50 cartucce a 1 mg DNA/0.25 mg oro / cartuccia. Ogni cartuccia è per un solo colpo. Le particelle d&#39;oro può essere sia 0,6 mm o 1,0 micron di diametro (Cat # 1652262 o Cat # 1652263; Biorad). Si consiglia inoltre di utilizzare 0,5 mg / ml soluzione PVP per la preparazione delle cartucce. Per BiFC, combinare le due DNA plasmide di ciascun partner interagire putativo nella preparazione stessa cartuccia. Dopo la preparazione delle cartucce, è importante per testare le cartucce da sparare le cartucce appena fatto ad una pressione di elio da 150 a 200 psi Parafilm in una piazza che è avvolto intorno ad una piastra di Petri. In questo modo verificare se le particelle d&#39;oro può essere azionato in modo efficace dalle cartucce, si dovrebbe vedere particelle d&#39;oro debole sul Parafilm. Questo vi dà un&#39;idea della zona che ogni colpo coprirà. Si possono notare diverse quantità di particelle d&#39;oro spinto sulla Parafilm a diverse pressioni di elio (a 150-200 gamma psi). Se non vi è alcuna differenza significativa, scegliere la pressione più bassa per il bombardamento. III. Tessuti cipolla Preparazione Il giorno di riprese, staccare la pelle esterna da una grossa cipolla gialla. Utilizzando una lama di rasoio pulito e affilato, tagliate 4-5 strati profondi un&#39;area rettangolare di circa 2 x 8 cm. Estrarre il tessuto rettangolare cipolla, scartare il due strati esterni, tagliate i restanti strati di cipolla a pezzi di circa 2x1.5 cm. Mettere i pezzi di cipolla in una capsula di Petri contenente carta da filtro (carta Whatman 3MM tagliata a cerchi) inumidito con acqua sterile. Coprire la piastra Petri e sono pronti a partire. IV. Bombardamento <ol><li> Caricare le cartucce nei supporti bianchi cartuccia rotonda. Cartucce contenenti diversi costrutti plasmide deve essere caricato in diversi titolari cartuccia. Ciascun titolare di cartuccia può contenere fino a 12 cartucce per 12 scatti. </li><li> Inserire la batteria e la fodera barile fresca nel Helios Gene Gun. Assicurarsi che sia il rivestimento canna e la pistola sono o-ring rispettivi allegati. </li><li> In primo luogo, inserire un supporto vuoto cartuccia nella pistola del gene, con il marchio # 12 sul supporto della cartuccia verso la parte superiore della pistola. Cartuccia titolare anticipo una o due volte per assicurarsi che sia inserita correttamente. </li><li> Fissare pistola al serbatoio di elio e regolare la pressione da 150 a 200 psi. </li><li> Indossare protezioni per le orecchie e pistola fuoco un paio di volte con il supporto della cartuccia vuota a ripulire la camera della pistola e per assicurarsi che la pressione è stabile. </li><li> Togliere il porta vuota della cartuccia e inserire un supporto caricato cartuccia. Anticipo il supporto della cartuccia in modo che la cartuccia da fuoco è posizionato in basso (06:00) del supporto della cartuccia. La ripresa di un pezzo di cipolla qui fornisce una forma di controllo negativo per fluorescenza di fondo. </li><li> Luogo pezzi di cipolla nel mezzo di un piatto vuoto Petri con lo strato più interno della cipolla rivolto verso l&#39;alto. Resto rivestimento canna del fucile gene su piastra di Petri e il fuoco della pistola tenendo premuto il pulsante laterale mentre si tira il grilletto. Trasferire la cipolla bombardato a una piastra di Petri contenente umido carta 3MM filtro. </li><li> Passare alla cartuccia successiva e fuoco un altro pezzo di cipolla. Ripetere questo passaggio fino a 4-5 pezzi di cipolle vengono uccisi. Ogni pezzo cipolla è sparato una sola volta. </li><li> Cambia per un supporto diverso cartuccia caricata con cartucce contenenti una diversa combinazione plasmide o diversi plasmide. Ricordatevi di cambiare il rivestimento barile unos anche per prevenire la contaminazione. Spara 4-5 pezzi di cipolla. </li><li> Dopo il bombardamento, restituire tutte le cipolle a piatti Perti. Coprire le piastre di Petri con coperchi. Avvolgere con Parafilm per evitare l&#39;essiccazione. Incubare i pezzi di cipolla a temperatura ambiente al buio per 16-48 ore. </li><li> Spegnere la pressione del gas e rilasciare prima di staccare la pistola gene dal serbatoio di elio. Svitare il rivestimento barile, e rimuovere il supporto della cartuccia e le cartucce utilizzate (o non utilizzate). </li><li> Pulizia titolari cartuccia e rivestimenti barile immergendo in un bicchiere con acqua saponata. Porre il becher in un bagno sonicatore e ultrasuoni per 20 minuti. Successivamente, sciacquare con acqua per eliminare tutti i residui di sapone. Ammollo in etanolo al 70% per un&#39;ora e poi asciugarle su carta assorbente. </li></ol> Osservazione V. <ol><li> Dopo l&#39;incubazione pezzi di cipolla bombardato per 16-20 ore a temperatura ambiente, si può osservare GFP o YFP fluorescenza utilizzando un microscopio a fluorescenza come il Axio Zeiss Observer.z1 utilizzato in questo studio. </li><li> Utilizzare pinze con estremità piatta lentamente buccia uno strato singola cellula fuori l&#39;epidermide interna della cipolla (il livello che si affaccia direttamente la pistola durante il bombardamento). Mettere la buccia su una goccia d&#39;acqua su un vetrino. Aggiungi coprioggetto ma attenzione a ridurre al minimo le bolle. </li><li> In campo chiaro del microscopio, primo controllo per lo streaming citoplasmatica per assicurare che le cellule sono vive. Per fare questo, esporre la diapositiva alla luce campo chiaro per alcuni minuti per riscaldare il tessuto cipolla e quindi cercare plastidi in movimento. Lo streaming citoplasmatica non è sempre facile da vedere, quindi non scoraggiatevi se non riesci a vedere nessuno. </li><li> Per il rilevamento a fluorescenza, l&#39;uso appropriato di eccitazione e filtri di rilevamento per GFP o YFP. E &#39;importante includere controlli negativi, come pezzi di cipolla girato con un non-fluorescente plasmide. Deboli segnali giallo potrebbe apparire dalle cellule feriti, bolle d&#39;aria, o detriti. E &#39;inoltre importante includere controlli positivi. Un plasmide contenente giornalista costitutivamente espresso fluorescenti come 35S:: GFP è un eccellente controllo positivo. Segnali GFP visibile dalle cellule epidermiche di cipolla (Figura 1A, D) ha indicato che le cartucce, i tessuti cipolla, e la pistola sparando gene siano adeguatamente preparati ed eseguiti. Successivamente, abbiamo osservato le interazioni tra SEU-pSPYNE e LUH-pSPYCE cipolla a pezzi bombardato con cartucce contenenti una miscela di 35S:: SEU-pSPYNE e 35S:: LUH-pSPYCE DNA. Debole fluorescenza YFP è stata osservata solo nel nucleo (Figura 1 B, E), indicando la diretta interazione fisica tra SEU e LUH che ha portato i frammenti YFP dividere insieme nel nucleo. Si noti che il segnale YFP da BiFC è significativamente più debole rispetto al segnale GFP da 35S:: GFP. </li></ol> Rappresentante Risultati Nel nostro studio ha riportato qui, il 35S:: GFP controllo positivo plasmide emana una forte fluorescenza che riempie l&#39;intera cella, inclusi sia il nucleo e il citoplasma (Figura 1A, D). Proteina GFP è abbastanza piccolo da diffondere in nucleo, senza un segnale di localizzazione nucleare. Al contrario, SEU e LUH sono due fattori di trascrizione di Arabidopsis precedentemente dimostrato di interagire in un doppio ibrido di lievito test 2. SEU-pSPYNE (SEU fuso al frammento N-terminale di YFP) e LUH-pSPYCE (SEU fuso al frammento N-terminale di YFP), sia troppo grande per diffondere nel nucleo, contengono segnali di localizzazione nucleare 2,5. Il segnale fluorescente YFP rilevato nel nucleo della cellula cipolla (Figura 1 B, E) indica che SEU-pSPYNE e LUH-pSPYCE possono interagire in nuclei cipolla. Nessun segnale fluorescente viene rilevato cipolle bombardati con mix di pSPYNE plasmide vettore plasmidico contenente il frammento N-terminale di YFP e LUH-pSPYCE (Figura 1C, F). Il BiFC-mediata fluorescenza YFP è significativamente più debole del fluorescenza GFP-mediata dal 35S:: GFP plasmide. Nel nostro studio, hanno anche mostrato diverse localizzazione subcellulare (cfr. Figura 1 A con B). Inoltre, il numero di cellule fluorescenti è significativamente inferiore per BiFC, come BiFC funziona solo quando entrambi i plasmidi partner di entrare con successo e sono espressi nelle cellule cipolla stesso. <img src="/files/ftp_upload/1963/1963fig1.jpg" alt="Figura 1" /> Figura 1. Fluorescente (A, B, C) ​​e in campo chiaro (D, E, F) le immagini di cellule di cipolla bombardati da diversi costrutti di plasmide. (A) e (D), le cellule Cipolla bombardati da 35S:: GFP. GFP diffonde attraverso il citoplasma e nucleo. (B) e (E), le cellule Cipolla bombardati con eguale mescola molare di 35S:: SEU-pSPYNE (SEU fuso al frammento N-terminale di YFP) e 35S:: LUH-pSPYCE (LUH fuso al frammento C-terminale ) DNA plasmidico. Un&#39;interazione tra SEU-pSPYNE e LUH-pSPYCE è indicato da un nucleo fluorescente. (C) e (F), un controllo negativo che mostra cellule cipolla bombardati da miscele di vettore pUC19-SPYNE e LUH-pSPYCE plasmids. Nessun segnale fluorescente viene rilevato.

<ol>
	<li>Hu, C. D., Chinenov, Y., 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+interactions+among+bZIP+and+Rel+family+proteins+in+living+cells+using+bimolecular+fluorescence+complementation.">Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.</a> Mol Cell. 9 (4), 789-789 (2002).</li><li>Sitaraman, J., Bui, M., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LEUNIG_HOMOLOG+and+LEUNIG+perform+partially+redundant+functions+during+Arabidopsis+embryo+and+floral+development.">LEUNIG_HOMOLOG and LEUNIG perform partially redundant functions during Arabidopsis embryo and floral development.</a> Plant Physiol. 147 (2), 672-672 (2008).</li><li>Walter, 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+protein+interactions+in+living+plant+cells+using+bimolecular+fluorescence+complementation.">Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation.</a> Plant J. 40 (3), 428-428 (2004).</li><li>Woods, G., Zito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+gene+gun+bullets+and+biolistic+transfection+of+neurons+in+slice+culture.">Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.</a> J Vis Exp. , (2008).</li><li>Azhakanandam, S., Nole-Wilson, S., Bao, F., Franks, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SEUSS+and+AINTEGUMENTA+mediate+patterning+and+ovule+initiation+during+gynoecium+medial+domain+development.">SEUSS and AINTEGUMENTA mediate patterning and ovule initiation during gynoecium medial domain development.</a> Plant Physiol. 146 (3), 1165-1165 (2008).</li><li>Zhang, X. N., Mount, 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=Two+alternatively+spliced+isoforms+of+the+Arabidopsis+SR45+protein+have+distinct+roles+during+normal+plant+development.">Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development.</a> Plant Physiol. 150 (3), 1450-1450 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

<ol><li> Per gli utenti prima volta, si consiglia di praticare l&#39;intera procedura, dalla preparazione delle cartucce di osservazione, con un singolo giornalista plasmide costitutivo come 35S:: GFP o 35S:: GUS. Abbiamo usato un 35S:: GFP plasmide ottenuto da Drs. Steve Monte e Xiaoning Zhang 6 (Figura 1A, D). Le cartucce colpo di controllo gene venduti a RioRad (Cat 165-2244) non sono adatti per le piante. </li><li> Per BiFC, un secondo controllo positivo è necessario, che dovrebbe consistere in due partner noto che interagiscono...

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<td>Yellow onion</td>
<td>&nbsp;</td>
<td>Any supermarket</td>
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<td>Helios Gene Gun</td>
<td>&nbsp;</td>
<td>BioRad</td>
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<td>Fluorescent microscope with appropriate filters for GFP or YFP</td>
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<td>Zeiss, Nikon, Olympus</td>
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bimolecular fluorescence complementation (bifc) assay for protein-protein interaction in onion cells using the helios gene gun

Indagine della funzione del gene in diversi organismi si basa sulla conoscenza di come i prodotti gene interagiscono tra loro nel loro ambiente normale cellulare. La fluorescenza complementazione bimolecolare (BiFC) Assay<sup&gt; 1</sup&gt; Permette ai ricercatori di visualizzare le interazioni proteina-proteina nelle cellule viventi ed è diventato uno strumento di ricerca fondamentale. Questo test si basa sulla associazione agevolata di due frammenti di una proteina fluorescente (GFP) che sono ogni fuso con un potenziale partner di proteine ​​interagenti. L&#39;interazione dei due partner proteina faciliterebbe l&#39;associazione dei N-terminale e C-terminale frammento di GFP, portando a fluorescenza. Per i ricercatori cellule vegetali, cipolla epidermiche sono un sistema ideale di sperimentazione per condurre l&#39;analisi BiFC a causa della facilità nel raccogliere ed elaborare i tessuti cipolla e la visualizzazione diretta della fluorescenza con fluorescenza di fondo minima. L&#39;Helios Gun Gene (BioRad) è comunemente usato per bombardare DNA plasmidico in cellule di cipolla. Dimostriamo l&#39;uso di Helios Gun Gene introdurre costruisce plasmide per due interagenti<em&gt; Arabidopsis thaliana</em&gt; Fattori di trascrizione, Seuss (SEU) e omologo Leunig (LUH)<sup&gt; 2</sup&gt; E la visualizzazione delle loro interazioni mediate da BiFC nelle cellule epidermiche di cipolla.

Watch this Scientific Journal Video about Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun at JoVE.com

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

In questo articolo viene illustrato come utilizzare correttamente il BioRad Helios Gene Gun per introdurre DNA plasmidico in cellule epidermiche di cipolla e come test per interazioni proteina-proteina in cellule di cipolla in base al principio della complementazione bimolecolare fluorescenza (BiFC)

Biomolecolare fluorescenza complementazione (BiFC) Assay per interazione proteina-proteina nelle cellule Cipolla Utilizzando la Helios Gun Gene

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

bimolecular-fluorescence-complementation-bifc-assay-for-protein

Research

JoVE Journal

Biology

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

19.5K Views.  University of Maryland. In questo articolo viene illustrato come utilizzare correttamente il BioRad Helios Gene Gun per introdurre DNA plasmidico in cellule epidermiche di cipolla e come test per interazioni proteina-proteina in cellule di cipolla in base al principio della complementazione bimolecolare fluorescenza (BiFC)

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

Gene-gun Transfection of Hippocampal Neurons

Gene-gun trasfezione di neuroni dell&#39;ippocampo

Ricerca

Biologia

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

Preparazione di proiettili pistola Gene e Biolistic Transfection dei neuroni in cultura Slice

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

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.

Bimolecolari complementazione fluorescenza

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

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.

Rilevamento delle interazioni proteiche in pianta utilizzando un gateway compatibile complementazione fluorescenza biomolecolare (BiFC) Sistema

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

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Un liquido fase di cattura test affinità utilizzando biglie magnetiche per studiare interazioni proteina-proteina: il poliovirus-Nanobody Esempio

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Utilizzando Sequence SECM arresto come uno strumento per isolare ribosoma Polipeptidi Bound

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

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.

L&#39;analisi citofluorimetrica di bimolecular Complementation Fluorescenza: A High Throughput Metodo quantitativo per studiare l&#39;interazione proteina-proteina

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.

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 ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Imaging di fluorescenza con One nanometri Precisione (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...