AF è stato sostenuto da una borsa di studio a partire dal Consiglio europeo della ricerca nell&#39;ambito del Settimo programma quadro della Comunità europea (FP7 / 2007-2013) / accordo di sovvenzione del CER n ° 203.413 e dal Centro Minerva per la Bio-ibrido systems.HA complesso e AI sono supportati dal Dalia e Dan Maydan Fellowship per gli studenti di laurea specialistica presso l&#39;Università Ebraica di Gerusalemme.

1. Progettazione di un array di peptidi <ol><li> Dividere la sequenza della proteina bersaglio in parte si sovrappongono 10-20 residui peptidi. Variare la quantità di sovrapposizione sulla esperimento specifico e le risorse del laboratorio esecuzione, ma in linea di principio più lunga è la sovrapposizione meglio. Nel progettare i peptidi tengono conto noto elementi della struttura secondaria della proteina che può essere responsabile per l&#39;interazione. </li><li> Ordinare l&#39;array peptide progettato presso i rivenditori commerciali. Qui, uso peptidi legati covalentemente alla matrice attraverso il C-terminale. </li></ol> 2. Blocco legame non specifico <ol><li> Effettuare una soluzione tampone Tris 50 mM o fosfato contenente 0,05% di Tween 20 (TBST / PBST) e regolare il pH desiderato con una quantità misurata di HCl o NaOH (per conoscere la forza ionica precisi) e la forza ionica desiderato con NaCl . Qui, utilizzare un pH di 7,5 ed una forza ionica di 150 mM. </li><li> Fare una soluzione di saturazione di2,5% (w / v) di latte scremato in polvere in TBST / PBST. </li><li> Per prevenire il legame non specifico, immergere la matrice in 5 ml di soluzione bloccante. Incubare la matrice per 2-4 ore a temperatura ambiente o per una notte a 4 ° C su un agitatore. </li></ol> 3. Incubare con la proteina <ol><li> Lavare la matrice prima con 5 ml di soluzione di blocco per 30 sec e poi due volte con 5 ml di TBST / PBST per 5 minuti su un agitatore a temperatura ambiente. </li><li> Incubare la matrice lavato con 5 ml di His-tag soluzione proteica contenente 2,5% (w / v) di latte scremato in polvere per impedire legame non specifico. Qui, utilizzare 4,5 mM di soluzione proteica (STIL 500-650) disciolto nella soluzione di blocco descritto. NOTA: Generalmente 5-10 mM proteine ​​sono utilizzati per lo screening, ma la concentrazione di proteina può essere ancora a partire da 2-3 micron, a seconda della affinità di legame e le concentrazioni locali di peptidi che delimitano sulla matrice (efficienza del sintesi). Il buffer in cui la proteinaviene disciolto può variare ma è comune per diluire la proteina utilizzando la stessa soluzione di saturazione sopra descritto. </li><li> Incubare la matrice nella soluzione proteica per 3-8 ore a temperatura ambiente o per una notte a 4 ° C. </li></ol> 4. Incubare con l&#39;anticorpo <ol><li> Lavare l&#39;array tre volte con 5 ml di TBST / PBST. Il primo lavaggio è di 30 secondi seguito da due lavaggi 5 min su agitatore a temperatura ambiente. </li><li> Incubare la matrice lavato con 5 ml di anticorpo HRP-coniugato diluito (1: 1.500) in un tampone di incubazione che contiene gli stessi ingredienti alle stesse concentrazioni come la soluzione di saturazione, per 1 ora su un agitatore a temperatura ambiente. L&#39;anticorpo può legarsi sia alla proteina bersaglio o il tag. </li><li> Eseguire esperimenti di controllo testare l&#39;interazione dell&#39;anticorpo con peptidi sull&#39;array, specialmente se l&#39;array contiene peptidi dalla proteina bersaglio (ad esempio, per oligomerizzazione studi) ripetendo lo stesso protocollo senza step 3, incubando con la proteina. </li><li> Lavare l&#39;array tre volte con 5 ml di TBST / PBST. Il primo lavaggio è di 30 secondi seguito da due lavaggi 5 min su agitatore a temperatura ambiente. </li></ol> 5. Lettura del Array <ol><li> Effettuare sviluppo chemiluminescenza con un kit di substrato western blotting ECL. </li><li> Eseguire il rilevamento con un analizzatore di immagini luminescenti. </li><li> Analizzare i risultati. Assicurarsi che entrambi i duplicati su matrice mostrano gli stessi segnali, quindi i risultati sono affidabili. Se la struttura del partner proteina è noto, la ricerca sulla struttura dei peptidi leganti e cercare i siti di legame. Anche i peptidi che sono molto nella sequenza possono essere vicini nella struttura terziaria e creare un sito di legame. </li></ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+microcephaly+protein+STIL+interacts+with+CPAP+and+is+required+for+procentriole+formation.">The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation.</a> Embo J. 30 (23), 4790-4804 (2011).</li><li>Vulprecht, J., David, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STIL+is+required+for+centriole+duplication+in+human+cells.">STIL is required for centriole duplication in human cells.</a> Journal of Cell Science. 125 (5), 1353-1362 (2012).</li><li>Campaner, S., Kaldis, P., Izraeli, S., Kirsch, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sil+phosphorylation+in+a+Pin1+binding+domain+affects+the+duration+of+the+spindle+checkpoint.">Sil phosphorylation in a Pin1 binding domain affects the duration of the spindle checkpoint.</a> Mol Cell Biol. 25 (15), 6660-6672 (2005).</li><li>Kasai, K., Inaguma, S., Yoneyama, A., Yoshikawa, K., Ikeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCL/TAL1+interrupting+locus+derepresses+GLI1+from+the+negative+control+of+suppressor-of-fused+in+pancreatic+cancer+cell.">SCL/TAL1 interrupting locus derepresses GLI1 from the negative control of suppressor-of-fused in pancreatic cancer cell.</a> Cancer Res. 68 (19), 7723-7729 (2008).</li><li>Chaturvedi, P., Sudakin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chfr+regulates+a+mitotic+stress+pathway+through+its+RING-finger+domain+with+ubiquitin+ligase+activity.">Chfr regulates a mitotic stress pathway through its RING-finger domain with ubiquitin ligase activity.</a> Cancer Res. 62 (6), 1797-1801 (2002).</li><li>Olaussen, K. a., Commo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+proapoptotic+effects+of+the+two+tyrosine+kinase+inhibitors+pazopanib+and+lapatinib+on+multiple+carcinoma+cell+lines.">Synergistic proapoptotic effects of the two tyrosine kinase inhibitors pazopanib and lapatinib on multiple carcinoma cell lines.</a> Oncogene. 28 (48), 4249-4260 (2009).</li><li>Reingewertz, T. 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The authors have nothing to disclose.

Lo screening matrice Peptide è un ottimo strumento per identificare i siti di legame di una proteina bersaglio nei suoi partner. Questo test è veloce e facile da eseguire e il risultato può essere ottenuto in uno o due giorni lavorativi. Proiezione matrice Peptide è versatile e può essere utilizzato per vari scopi. Può essere usato per caratterizzare modifiche Traduzione successive come fosforilazione e identificare substrati di chinasi. Ad esempio: la chinasi FLT3, che è legato alla leucemia mieloide acuta, fosf...

Le interazioni proteina-proteina mediano maggior parte dei processi nella cellula vivente. Proteina deteriorate possono provocare malattie, rendendo le interazioni proteina importanti bersagli farmacologici. È quindi estremamente importante capire queste interazioni a livello molecolare. Interazioni proteine ​​sono studiate utilizzando una varietà di tecniche che vanno da saggi cellulari e biochimici per saggi biofisici quantitative, e questi possono essere eseguite sia con proteine ​​integrali, con domini proteici o peptidi. Peptidi servono come ottimi strumenti per studiare le interazioni delle proteine. Questo perché i peptidi possono essere facilmente sintetizzati e consentono la messa a fuoco su un sito di interazione specifica su un lato e più bersagli proteici in maniera elevata produttività dall&#39;altro 1,2 mano. Proiezione matrice Peptide è un veloce, facile da eseguire metodo per ottenere una grande quantità di dati sulle interazioni di una proteina bersaglio con numerosi partner in breve tempo 3. A differenza di altri metodi biochimici o biofisici per la rilevazione e l&#39;analisi di interazioni proteina-proteina, lo screening matrice peptide richiede una concentrazione molto bassa di proteine ​​e può rilevare molto debole legame. Array di peptidi possono essere utilizzati per molte applicazioni nelle interazioni peptide-proteina come la mappatura della proteina-proteina o recettore-ligando siti di interazione 4, interfacce omo- o etero-oligomerizzazione, anticorpi che caratterizzano epitopi 5, studiando attività enzimatiche 6 e throughput elevato struttura- rapporto di attività (SAR) studia 7. Per un esame approfondito circa lo screening gamma peptide vedere Katz et al. 4 Attualmente esistono diversi tipi di matrici di peptidi. Ci sono due principali strategie sintetiche per la preparazione di matrici peptide: sintesi dei peptidi prima di essere esposti al supporto solido, o per la sintesi di peptidi direttamente sul supporto solido, principalmente con la tecnica SPOT 4,8. Lapeptidi sono sintetizzati sul supporto solido solito da 9-fluorenilmetilossicarbonil (Fmoc) chimica 8. Tra gli schemi sintetici comuni sono allegati peptide attraverso il terminale N (ad esempio, JPT array pepstar 9) e l&#39;attaccamento peptide attraverso il terminale C (ad esempio, PEPSCAN array pepchip 10, JPT array pepspot 9 e INTAVIS array celluspot 11) 4. Il supporto solido può variare e così fa la chimica dell&#39;accoppiamento peptide ad esso. Peptidi Cys-terminati possono essere allegati ai vetrini tramite il gruppo tiolo 12. Il terminale N di un peptide può essere legato covalentemente ad un gruppo ossidrile sulla membrana di cellulosa attraverso esterificazione degli aminoacidi allegato 8. Qui vi presentiamo un protocollo dettagliato per lo screening array di peptidi come metodo per lo studio delle interazioni proteina-proteina. L&#39;array che abbiamo usato è l&#39;array Celluspots, che è un micro-array che contiene grandi amount di macchie (duplicati fino a 384 punti) su una piccola membrana di cellulosa supportati da un vetrino. In questo modo si lavora con bassi volumi di proteine ​​e anticorpi e di ottenere significative quantità di dati per ogni singolo esperimento. Questa matrice contiene anche ad alta densità peptide che permette la rilevazione di bassa affinità di legame. L&#39;array è stato utilizzato per mappare l&#39;interazione STIL-CHFR, che è molto importante per il controllo della proliferazione delle cellule normali 13. Interazione incontrollata tra le due proteine ​​può portare allo sviluppo del cancro. Con la mappatura questa interazione abbiamo trovato il sito di legame specifico e residui 14 vincolanti. Questo spiana la strada per la progettazione di inibitori razionali che inibiscono questa interazione proteina-proteina.

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			<td height="21" style="height:21px;width:231px;">Name of Reagent/ Equipment</td>
			<td style="width:231px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;">Peptide array</td>
			<td>INTAVIS Bioanalytical Instruments</td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">His-probe Antibody (H-3) HRP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8036 HRP</td>
			<td></td>
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			<td height="21" style="height:21px;">EZ-ECL Kit</td>
			<td>Biological industries</td>
			<td>20-500-120</td>
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			<td height="21" style="height:21px;">Skim Milk</td>
			<td>Becton, Dickinson and Company</td>
			<td style="width:119px;">232100</td>
			<td></td>
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			<td height="21" style="height:21px;">LAS-3000 camera&#160;</td>
			<td>FUJI film</td>
			<td></td>
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identifying protein-protein interaction sites using peptide arrays

Le interazioni proteina-proteina mediano maggior parte dei processi nella cellula e soggiorno di controllo omeostasi dell&#39;organismo. Proteina deteriorate possono provocare malattie, rendendo le interazioni proteina importanti bersagli farmacologici. È quindi estremamente importante capire queste interazioni a livello molecolare. Interazioni proteine ​​sono studiate utilizzando una varietà di tecniche che vanno da saggi cellulari e biochimici per saggi biofisici quantitative, e questi possono essere eseguite sia con proteine ​​integrali, con domini proteici o peptidi. Peptidi servono come ottimi strumenti per studiare le interazioni proteina da peptidi possono essere facilmente sintetizzati e consentire la messa a fuoco su siti specifici di interazione. Array peptidici permettono l&#39;identificazione dei siti di interazione tra due proteine ​​così come screening per peptidi che si legano alla proteina bersaglio per scopi terapeutici. Essi consentono inoltre ad alto rendimento studi SAR. Per l&#39;identificazione di siti di legame, un Typimatrice peptide cal solito contiene parzialmente sovrapposte 10-20 residui peptidi derivati ​​dalle sequenze complete di una o più proteine ​​partner della proteina bersaglio desiderato. Screening matrice per legare la proteina bersaglio rivela i peptidi leganti, corrispondenti ai siti di legame delle proteine ​​partner, in un modo semplice e veloce utilizzando solo piccole quantità di proteine. In questo articolo si descrive un protocollo per lo screening array di peptidi per la mappatura dei siti di interazione tra una proteina bersaglio e dei suoi partner. L&#39;array peptide è stato progettato sulla base delle sequenze delle proteine ​​partner, tenendo conto delle loro strutture secondarie. Gli array utilizzati in questo protocollo sono stati Celluspots array preparati da INTAVIS bioanalitici Instruments. L&#39;array è bloccato per impedire il legame aspecifico e quindi incubate con la proteina studiata. Rilevamento utilizzando un anticorpo rivela i peptidi di legame corrispondenti ai siti specifici di interazione tra le proteine.

STIL è una proteina molto importante centrosomica. Controlla normale divisione cellulare e la proliferazione delle cellule 13,15 - 19. STIL interagisce con numerose proteine ​​18,20,21, e la maggior parte delle interazioni si verificano attraverso la sua parte centrale, che è una regione intrinsecamente disordinati (IDR) 14. Abbiamo progettato un array costituito da peptidi derivati ​​dalla proteina legante STIL CHFR. CHFR è un soppressore del tumore che è indotta in risposta...

Watch this Scientific Journal Video about Identifying Protein-protein Interaction Sites Using Peptide Arrays at JoVE.com

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Identificare proteina-proteina siti di interazione Uso Peptide Array

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

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17.9K Views.  The Hebrew University of Jerusalem. Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Video: Identifying Protein-protein Interaction Sites Using Peptide Arrays

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 ...

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Biologia

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)

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 ...

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, ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenesi e Analisi funzionale dei canali ionici Heterologously Espresso in cellule di mammifero

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

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 ...

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 ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Identificare proteina-proteina interazione in<em&gt; Drosophila</em&gt; Adult capi di Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

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 ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

Analisi Specificità di proteine ​​lisina metiltransferasi Utilizzando SPOT Peptide Array

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Genome-wide proteina-proteina interazione Screening per Protein-frammento Complementation Assay (PCA) in cellule viventi

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...