This work was supported by a grant from the NIH. The assays shown in Figure 1 was performed by Dr. Joseph Fasolo. We thank Dr. Rui Chen for helpful comments.

1. Preparazione della sonda <ol><li> Cultura e purificare le sonde chinasi V5-fusione utilizzate per esaminare le interazioni con altre proteine ​​come segue: <ol><li> Utilizzare appena striato ceppo di lievito Y258 (Mata pep4-3, his4-580, ura3-53, leu2-3,112) contenenti proteine ​​V5-fusion (espressa da GATEWAY vettoriali pYES-DEST52). Utilizzare la proteina isolata come sonda sui microarray. Piatto il lievito in sintetico complete-uracile (Sc-Ura) / 2% destrosio / Agar e crescere a 30 ° C per 3 giorni di cultura congelati (-80 ° C glicerolo magazzino). </li><li> Seminare colture starter (5-20 ml) da una singola colonia e crescere durante la notte in Sc-Ura / 2% destrosio sulla piattaforma (220-250 giri al minuto) o la rotella che agita a 30 ° C. </li><li> La mattina seguente, inoculare 400 ml di Sc-Ura / 2% cultura raffinosio con la cultura di avviamento sufficiente per un diametro finale 600 del 0.1. </li><li> Crescere i inoculums a OD 600 di 0,6, seguita da induzione galattosio del fu V5-chinasisione costruire espressione aggiungendo una soluzione di 3x Yeast Extract / Peptone (YEP) supplementato con 6% galattosio, aggiungendo abbastanza per diluire il supporto di induzione di un fattore 3 in modo che la concentrazione finale di galattosio è 2%. </li><li> Indurre le cellule a 30 ° C per 6 h su una piattaforma oscillante. Utilizzare un pallone da 2 L Erlenmeyer per garantire un&#39;adeguata aerazione. </li><li> Celle di raccolta utilizzando un rotore JA-10 (o comparabili) facendo girare 400 ml di sospensione cellulare a 1000 xg per 5 minuti a 4 ° C. </li><li> Lavare le cellule una volta con 50 ml di ghiaccio tampone PBS freddo e trasferirlo in una provetta da 50 ml. Lavare il pellet di nuovo in ghiaccio PBS freddo (senza detergenti o altri additivi) utilizzati per lisi e trasferimento in 2 ml snap-cap tubi per lisi. </li><li> Spin le cellule a 20.000 xg per 1 min a 4 ° C per un pellet e pipetta via buffer. </li><li> Mettere le provette su ghiaccio e procedere con il passaggio di lisi. </li><li> Lyse le cellule (250-350 ml pellet) con 00,5 millimetri perle zirconia in un 1: 1: 1 volume di pellet, perline, e PBS (PBS) Lysis Buffer e vortex la miscela utilizzando una piattaforma di agitazione per 3 volte a 2 min a intervalli di 4 ° C. </li><li> Centrifugare il lisato a 20.000 xg in una microcentrifuga da tavolo per 10 minuti a 4 ° C. </li><li> Dispensare surnatante in provetta da centrifuga in policarbonato ad alta velocità e chiarire da ulracentrufugation per 30 min a 150.000 xg a 4 ° C. </li><li> Trasferire il lisato chiarificato in una provetta contenente prelavato Ni 2 + resina di affinità (~ 100 ml) e incubare su un nutator per 2 ore a 4 ° C per catturare istidina (His) 6X tagged proteine ​​V5-fusion. </li><li> Lavare la resina tre volte per 10 minuti a 4 ° C con tampone di lavaggio. Pellet resina utilizzando una centrifuga da tavolo per 5 min a 1000 xga 4 ° C e aspirare il surnatante. Aggiungere tampone di lavaggio fresco per ogni lavaggio e restituire il tubo del nutator per l&#39;agitazione durante il lavaggio. </li><li> Dopo il lavaggio steps sono completi, applicare la resina lavato per un nuovo G-25 colonna per eluizione. </li><li> Applicare 25 microlitri di tampone di eluizione in maniera graduale iniziando con imidazolo 100 mM imidazolo 500 mM in incrementi di 50 mm per un totale di 9 frazioni. </li><li> Analizzare frazioni raccolte utilizzando gel di sodio dodecil solfato poliacrilammide (SDS-PAGE) e colorazione Coomassie identificare la frazione (s) più altamente arricchito per l&#39;uso nel saggio e aggiungere glicerolo al 30% e conservare a -80 ° C fino all&#39;uso nel test. </li></ol></li></ol> 2. Probing gli Array NOTA: Si prega di vedere il punto 2.6 di questa sezione per preparare la soluzione di anticorpi prima di iniziare il test. <ol><li> Per rilevare le interazioni, diluire l&#39;anticorpo coniugato V5-fluoroforo (cioè AlexaFluor 647) a 260 ng / ml in tampone sonda e mescolare accuratamente agitando. NOTA: Preparare questa soluzione di anticorpo 30 min prima dell&#39;uso e porre il tubo su una nutato o ruota per permettere una miscelazione completa e una sospensione omogenea di anticorpi. </li><li> Diluire la sonda proteica V5-fusione su un intervallo di concentrazione di 5-500 mg / ml. NOTA: Ottimizzato per ogni proteina-proteina test interazione, utilizzando tampone sonda. Ottimizzazione comporta l&#39;aggiunta più sonde per sondare buffer. Tipicamente 10 ug / ml viene usata come punto di partenza e regolati di conseguenza sulla base della forza del segnale. </li><li> Rimuovere i microarrays della proteina dal congelatore (-20 ° C) e portare a 4 ° C in frigorifero immediatamente prima dell&#39;uso. </li><li> Aggiungere tampone di bloccaggio direttamente al titolare diapositiva contenente i micorarrays proteine ​​e coprire la parte superiore con parafilm per evitare perdite. Bloccare gli array in tampone bloccante per 1 ora agitando a 50 giri su un palco a 4 ° C. </li><li> Dopo il blocco, trasferire gli array ad una camera umidificata refrigerate a 4 ° C e aggiungere 90 ml di sonda diluito direttamente sulla superficie dell&#39;array. Sovrapporre il arrays con una scivolata sollevatore alzato e incubare statico (senza agitando) nella camera umidificata a 4 ° C per 1,5 ore. </li><li> Lavare gli array 3 volte per 1 min ciascuna in tampone sonda in tre provette da 50 ml coniche. Aggiungere la diapositiva per provette coniche contenenti abbastanza tampone sonda pre-raffreddata a busta completamente la diapositiva. Lasciare che la polizza sollevatore di scivolare delicatamente fuori del microarray di proteine ​​(non forzare fuori come questo potrebbe causare danni alla superficie array). </li><li> Applicare la soluzione di anticorpi direttamente a matrice immediatamente dopo aver completato il lavaggio (passo 2.5) e overlay con una scivolata sollevatore cresciuto come prima. Incubare le matrici per 30 min a 4 ° C in camera umidificata. </li><li> Eseguire la stessa fase di lavaggio come prima (3 volte 1 min in tampone sonda), e centrifuga in una provetta conica da 50 ml a 800 xg in una centrifuga da tavolo per 5 min a temperatura ambiente. Asciugare gli array in un supporto di diapositive al buio per 30 minuti prima scansione della matrice a 647 nm. </li></ol>

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The authors declare that they have no competing financial interests.

Il protocollo è stato originariamente presentato effettuata utilizzando 85 proteine ​​del lievito proteine ​​chinasi-V5 fusione unica di confrontare attività di legame attraverso distinte famiglie e connessi di protein chinasi lievito conseguente individuazione di nuove reti di interazione in vivo chinasi 1. Come un emergente tecnologia profilazione proteomica, lo sviluppo di High Throughput (HTP) proiezione del proteoma usando microarrays della proteina invocato librerie peptidiche e eucar...

La necessità di effettuare analisi globali di biochimica delle proteine ​​e di attività in vivo legame ha portato allo sviluppo di nuovi metodi di profiling interazioni proteina-proteina (PPI) e le modificazioni post-traduzionali di proteomi interi 1,3-8. Microarrays proteine ​​sono prodotte come microarray proteici funzionali utilizzando proteine ​​funzionali full-length 4-6,8,9 o microarrays della proteina analitiche contenenti anticorpi 10,11. Essi sono progettati per contenere una alta densità di proteine ​​disposte su vetrini da microscopio con una varietà di chimiche superficiali per facilitare una varietà di condizioni sperimentali necessari per condurre ampio biochimico analisi 12. Nitrocellulosa e di superficie aldeide chimiche per il fissaggio chimico tramite lisina o attaccamento affinità metodi come diapositive chelati nichel per fissare le proteine ​​His-tag e glutatione per il fissaggio affinità tra gli altri 13. </p&gt; L&#39;uso di microarray della proteina funzionali per rilevare le interazioni proteina-proteina richiede l&#39;accesso a una elevata qualità library proteina funzionale 14. S. cerevisiae è suscettibile di produrre una tale biblioteca attraverso l&#39;abbinamento di alta copia affinità tagged costrutti di proteine ​​ad alto throughput tecniche di purificazione cromatografica. La stragrande maggioranza del genoma del lievito è stato sequenziato e quasi l&#39;intero proteoma può essere espresso da un plasmide alta copia per la purificazione e biochimico analisi 12. Una volta che le proteine ​​sono ottenuti e disposte in formato da 384 pozzetti, vengono stampate su un vetrino da microscopio che permette una rapida analisi biochimica multi-parametrica parallelo e interrogatori bioinformatica 8,14-16. Microarrays della proteina sono stati utilizzati per saggi enzimatici e interazioni con proteine, lipidi, piccole molecole, e acidi nucleici tra molte altre applicazioni. L&#39;accessibilità delle proteine ​​sulla superficie del proteomaarray li rendono suscettibili di diversi tipi di rilevazione analitica, tra cui, immuno-affinità, risonanza plasmonica di superficie, fluorescenza e molte altre tecniche. Inoltre, esso consente un controllo preciso della condizione sperimentale dove potrebbe essere difficile da fare in vivo. L&#39;obiettivo di questo protocollo è quello di dimostrare l&#39;uso appropriato di microarrays della proteina funzionali per rilevare le interazioni proteina-proteina. Questa applicazione consente l&#39;analisi biochimica in parallelo ad alta produttività delle attività di legame alle proteine ​​usando un analita altamente purificato (proteina) di interesse. Una C-terminale (carbossi-terminale) etichettato V5-fusione esca proteina di interesse è prodotta da un plasmide alta copia in un ceppo di lievito ottimizzato per la purificazione della proteina. C-terminale codifica assicura che l&#39;intera lunghezza proteina è stato tradotto. La proteina utilizzata in questo studio è TDA1-V5 proteina di fusione chinasi, che viene potabilizzata con resina nichel affinità tramite un tag His6X. La costru fusione TDA1-V5t è purificato tramite eluizione seriale utilizzando un gradiente di imidazolo per eluire la frazione più altamente arricchito per l&#39;uso nel saggio.

<table><tbody><tr><td>Petri Dishes</td><td>VWR</td><td>NC-10747</td><td>or comparable</td></tr><tr><td>Bacto yeast extract</td><td>Difco</td><td>0217-17</td><td></td></tr><tr><td>Bacto peptone</td><td>Difco</td><td>0118-17</td><td></td></tr><tr><td>Bacto agar</td><td>Difco</td><td>0140-01</td><td></td></tr><tr><td>Dextrose</td><td>Sigma</td><td>D9434</td><td></td></tr><tr><td>Raffinose</td><td>Sigma</td><td>R0514</td><td></td></tr><tr><td>Galactose</td><td>Sigma</td><td>G0750</td><td></td></tr><tr><td>Sc-Ura drop out media</td><td>MP Bio</td><td>114410622</td><td></td></tr><tr><td>Small Culture tubes</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>Large Erlenmeyer Flask</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>Centrifuge JA-10 rotor</td><td></td><td></td><td></td></tr><tr><td>Centrifuge tubes (500 ml)</td><td></td><td></td><td></td></tr><tr><td>Falcon tube (50 ml)</td><td>VWR/Fisher</td><td>21008-951</td><td></td></tr><tr><td>PBS Tablets</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>FastPrep Tubes (2 ml screw cap tube)</td><td></td><td></td><td></td></tr><tr><td>FastPrep Machine</td><td>MP Biomedicals</td><td>116004500</td><td></td></tr><tr><td>Zircon Beads</td><td>BioSpec</td><td>11079105</td><td></td></tr><tr><td>Triton X100</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D9779</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S3014&amp;nbsp;</td><td></td></tr><tr><td>Imidazole (pH = 7.4)</td><td>Sigma</td><td>I5513&amp;nbsp;</td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td>93482</td><td></td></tr><tr><td>Complete Inhibitor Cocktail (EDTA Free)</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>Phosphatase inhibitor cocktail 1</td><td>sigma</td><td>P2850</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153&amp;nbsp;</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P1379&amp;nbsp;</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A1852&amp;nbsp;</td><td></td></tr><tr><td>Shaking Incubator (30 &amp;deg;C)</td><td></td><td></td><td></td></tr><tr><td>Nickel Affinity Resin</td><td>Life Technologies</td><td>R901-01</td><td></td></tr><tr><td>G25 column</td><td>GE life sciences</td><td>27-5325-01&amp;nbsp;</td><td></td></tr><tr><td>Nutator</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>SDS-PAGE Gel (NuPage)</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Coomassie Blue Stain</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Monoclonal V5 Antibody</td><td>Life Technologies</td><td>R960-25</td><td></td></tr><tr><td>Alexa647 Tagged monoclonal V5 Antibody</td><td>Life Technologies</td><td>451098</td><td></td></tr><tr><td>Protein Microarrays Kit</td><td>Life Technologies</td><td>PAH0525013</td><td></td></tr><tr><td>Genepix Scanner</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Lifter Slips</td><td>Thomas Scientific</td><td></td><td>http://www.thomassci.com/Supplies/Microscope-Cover-Glass/_/LIFTER-SLIPS/</td></tr><tr><td>Lysis Buffer: 0.1% Triton X-100, 0.5 mM DTT,&lt;br/&gt; 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 500 mM NaCl, 50 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &amp;ndash; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF&amp;nbsp; (add just prior to use)</td><td></td><td></td><td>Add the reagents to 1x PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride; pH 7.4) to obtain the desired volume of lysis solution</td></tr><tr><td>Blocking Buffer: 1% bovine serum albumin (BSA), 0.1% Tween-20</td><td></td><td></td><td>Add the reagents to 1x PBS to obtain the desired volume of blocking buffer</td></tr><tr><td>Probe Buffer: 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 0.5 mM DTT, 0.05% Triton X-100, 50 mM NaCl, 500 &amp;mu;M ATP (for kinases), 1% BSA</td><td></td><td></td><td>Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>Wash Buffer: 0.1% Triton X-100, 500 mM NaCl, 0.5 mM DTT, 50 mM imidazole (pH 7.4),2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>Elution Buffer: 0.1% Triton X-100, 0.5 mM DTT, 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 500 mM NaCl, 50 &amp;ndash; 500 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &amp;ndash; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF (add just prior to use)</td><td></td><td></td><td>Before you begin, it is important to note the concentration gradient of imidazole (50 &amp;ndash; 500 mM) and to create separate tubes for each concentration (it is advisable to create the interval using 50 mM increments). Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>3x YEP +6% galactose: Dissolve 30 g of yeast extract and&amp;nbsp; 60 g of peptone in 700 ml of H2O, and autoclave. Add 300 ml of filter sterilized 20% galactose (wt/vol)</td><td></td><td></td><td>&amp;nbsp;Galactose should not be autoclaved</td></tr></tbody></table>

probing high-density functional protein microarrays to detect protein-protein interactions

Ad alta densità di microarrays della proteina funzionali contenenti ~ 4.200 proteine ​​ricombinanti di lievito vengono esaminati per le interazioni proteina-proteina chinasi utilizzando una proteina di fusione purificate per affinità lievito chinasi contenente un tag V5-epitopi per read-out. Chinasi purificata è ottenuto attraverso la cultura di un ceppo di lievito ottimizzato per alta copia la produzione di proteine ​​ospitare un plasmide contenente una fusione chinasi-V5 costruire sotto un elenco indirizzi globale promotore inducibile. Il lievito viene coltivato in mezzi restrittiva con una fonte di carbonio neutro per 6 ore seguita da induzione con 2% galattosio. Successivamente, la coltura è raccolta e chinasi è purificato mediante affinità standard di tecniche cromatografiche per ottenere una proteina chinasi altamente purificata per uso nel saggio. La chinasi purificata è diluita con tampone chinasi ad una gamma appropriata per il dosaggio e microarray della proteina sono bloccate prima ibridazione con il microarray della proteina. Dopo l&#39;ibridazione, gli array vengono sondati con un anticorpo monoclonale V5 ANTIBody per identificare le proteine ​​legate dalla proteina chinasi-V5. Infine, gli array vengono sottoposti a scansione utilizzando uno scanner microarray standard ei dati vengono estratti per l&#39;informatica a valle dell&#39;analisi 1,2 per determinare una elevata fiducia insieme di interazioni proteiche per la convalida a valle in vivo.

L&#39;attività di interazione proteina-proteina è stata osservata utilizzando un lettore di chip standard per valutare l&#39;TDA1-V5 proteina chinasi di fusione costrutto come proteina esca contro un lievito microarray proteina funzionale contenente circa 4,200 unica S. cerevisiae proteine ​​di fusione GST-. Ulteriore interrogatorio con il software GenePix rivelato una moltitudine di eventi di intensità variabili vincolanti. L&#39;affinità è stato misurato l&#39;intensità graduata del segnale derivato...

Watch this Scientific Journal Video about Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions at JoVE.com

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

Utilizzando microarray proteici contenenti quasi tutta la S. cerevisiae proteoma è sondato per un rapido interrogatori imparziale di migliaia di interazioni proteina-proteina in parallelo. Questo metodo può essere utilizzato per la proteina-piccola molecola, modificazione post-traslazionale, e altri saggi 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 ...

probing-high-density-functional-protein-microarrays-to-detect-protein

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.0K Views.  Stanford University.  Utilizzando microarray proteici contenenti quasi tutta la S. cerevisiae proteoma è sondato per un rapido interrogatori imparziale di migliaia di interazioni proteina-proteina in parallelo. Questo metodo può essere utilizzato per la proteina-piccola molecola, modificazione post-traslazionale, e altri saggi in high-throughput. 

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

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Tecnologia di Microarray di proteina extracellulare per il rilevamento di Throughput elevato di interazioni recettore-ligando bassa affinità

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Ricerca

Biochimica

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Un approccio comparato alla caratterizzano il paesaggio di ospite-patogeno interazioni proteina-proteina

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Immunologia e infezioni

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

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

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

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

In vivo 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 ...

Biologia

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Anticorpo Microarray chimicamente bloccato per Multiplexed high-throughput Profiling di glicosilazione delle proteine ​​specifico in campioni complessi

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

Scoprire le interazioni proteina e caratterizzazione di proteine ​​Funzione Con HaloTag Tecnologia

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

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 ...

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 ...

Rilevamento ad alta sensibilità del legame composto-proteina mediante fluorescenza

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. Umbelliferone, an active ingredient in Tibetan medicine Vicatia thibetica, exhibits an immunomodulatory effect with an unknown mechanism. The CD40 protein is a key target in the immune response. Therefore, this study employs the principle of differential scanning fluorescence technology to analyze the interactions between CD40 protein and umbelliferone using fluorescent enzyme markers. Initially, the stability of the protein fluorescent orange dye was experimentally verified, and the optimal dilution ratio of 1:500 was determined. Subsequently, it was observed that the temperature melting (Tm) value of CD40 protein tended to decrease with an increase in concentration. Interestingly, the interaction between CD40 protein and umbelliferone was found to enhance the thermal stability of CD40 protein. This study represents the first attempt to detect the binding potential of small molecule compounds and proteins using fluorescence microplates and fluorescent dyes. The technique is characterized by high sensitivity and accuracy, promising advancements in the fields of protein stability, protein structure, and protein-ligand interactions, thus facilitating further research and exploration.

Autore in primo piano: Semplificazione dei metodi PCR per una maggiore accessibilità ed efficienza

This protocol describes a unique method for detecting the binding between compounds and protein molecules, offering the advantages of minimal protein sample loss and high data accuracy.

Rilevamento dell'interazione proteina-umbelliferone CD40 tramite fluorescenza a scansione differenziale

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. ...

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

Riepilogo

Esplora Altri Video

Capitoli in questo video

Tecnologia di Microarray di proteina extracellulare per il rilevamento di Throughput elevato di interazioni recettore-ligando bassa affinità

Un approccio comparato alla caratterizzano il paesaggio di ospite-patogeno interazioni proteina-proteina

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

Anticorpo Microarray chimicamente bloccato per Multiplexed high-throughput Profiling di glicosilazione delle proteine specifico in campioni complessi

Scoprire le interazioni proteina e caratterizzazione di proteine Funzione Con HaloTag Tecnologia

Identificare proteina-proteina siti di interazione Uso Peptide Array

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

Rilevamento dell'interazione proteina-umbelliferone CD40 tramite fluorescenza a scansione differenziale

Rilevamento dell'interazione proteina-umbelliferone CD40 tramite fluorescenza a scansione differenziale

Rilevamento dell'interazione proteina-umbelliferone CD40 tramite fluorescenza a scansione differenziale

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

Riepilogo

Esplora Altri Video

Capitoli in questo video

Tecnologia di Microarray di proteina extracellulare per il rilevamento di Throughput elevato di interazioni recettore-ligando bassa affinità

Un approccio comparato alla caratterizzano il paesaggio di ospite-patogeno interazioni proteina-proteina

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

Anticorpo Microarray chimicamente bloccato per Multiplexed high-throughput Profiling di glicosilazione delle proteine ​​specifico in campioni complessi

Scoprire le interazioni proteina e caratterizzazione di proteine ​​Funzione Con HaloTag Tecnologia

Identificare proteina-proteina siti di interazione Uso Peptide Array

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

Rilevamento dell'interazione proteina-umbelliferone CD40 tramite fluorescenza a scansione differenziale

Rilevamento dell'interazione proteina-umbelliferone CD40 tramite fluorescenza a scansione differenziale

Rilevamento dell'interazione proteina-umbelliferone CD40 tramite fluorescenza a scansione differenziale

Anticorpo Microarray chimicamente bloccato per Multiplexed high-throughput Profiling di glicosilazione delle proteine specifico in campioni complessi

Scoprire le interazioni proteina e caratterizzazione di proteine Funzione Con HaloTag Tecnologia