Gli autori ringraziano il personale del dipartimento di Biotecnologie Farmaceutiche e Biologia Molecolare e soprattutto De Monique Pelsmacker per la preparazione del virus radioattivo marcato. Siamo grati a Ellen Merckx e Hadewych Halewyck per le loro osservazioni e discussioni interessanti e di Gerrit De Bleeser per il suo aiuto in laboratorio. Questo lavoro è stato finanziato da una sovvenzione OZR della Vrije Universiteit Brussel (OZR1807), il Fonds voor Onderzoek Wetenschappelijk Vlaanderen (G.0168.10N) e l&#39;Organizzazione Mondiale della Sanità (TSA 200.410.791). Lise Schotte è un collega predoctoral del Fonds voor Onderzoek Wetenschappelijk Vlaanderen (FWO).

Il principio (A) e una panoramica del metodo (B) sono rappresentati nella Figura 1. 1. Preparazione del tampone <ol><li> Preparare Binding / tampone di lavaggio sciogliendo sodio diidrogeno fosfato (50 mM) e cloruro di sodio (300 mM) in acqua e regolare il pH a 8,0. Inoltre aggiungere Tween 20 (0,01% (m / v) concentrazione finale), metionina (2% (m / v) concentrazione finale) e albumina (0,1% (m / v), concentrazione finale) e regolare il volume desiderato. </li></ol> 2. Preparazione delle biglie magnetiche Preparare le biglie magnetiche in base al manuale di istruzioni. In breve: <ol><li> Risospendere le biglie magnetiche utilizzando un vortice. </li><li> Trasferimento, per ciascun campione, 10 microlitri delle microsfere in sospensione magnetiche (40 mg / ml) in una provetta. </li><li> Raccogliere le biglie magnetiche con un magnete, finché il surnatante è chiaro (+ / - 30 sec) e rimuovere il supernatante attentamente. </li><li> Lavare ilbiglie magnetiche due volte: risospendere le sfere in 150 microlitri di Binding / tampone di lavaggio. Migrazione le biglie magnetiche ad un lato del tubo utilizzando un magnete finché il surnatante è chiara e rimuovere il supernatante. </li><li> Uso 40 microlitri di Binding / tampone di lavaggio, per ciascun campione, per risospendere le sfere (10 mg / ml). </li></ol> 3. Affinity Capture Assay Nota: Per misurare il (cosmica) radioattività di fondo (0% della radioattività), nessun virus radiomarcato viene aggiunto a un campione di controllo 1. Invece, lo stesso volume Binding / lavaggio tampone viene aggiunto. Nota: 100% radioattività è definita da un 2 campione di controllo a cui vengono aggiunti tutti i componenti eccetto il nanobody. Invece, lo stesso volume Binding / lavaggio tampone viene aggiunto. <ol><li> Portare 2000 cpm di 35 S etichettati (β-radiazioni) poliovirus di tipo Sabin ceppo 1 9, diluito con Binding / Wash buffer per 80 microlitri in una piastra a 96 pozzetti microtiter. </li><li> Aggiungere 10 &amp; mu; l di diluizione nanobody ai pozzetti. </li><li> Mescolare per circa 10 secondi con un agitatore. </li><li> Permettere ai campioni di incubare per 1 ora a temperatura ambiente. </li><li> Aggiungere 40 microlitri della sospensione magnetica lavato perline e incubare per 10 minuti a temperatura ambiente, sotto continua agitazione. </li><li> Separate le perline e il supernatante utilizzando un magnete e trasferire il surnatante eliminato in un tubo. </li></ol> 4. Misurazione della radioattività <ol><li> Trasferire 50 pl del supernatante dal passaggio 3,6 in un pallone di conteggio. </li><li> Aggiungere 3 ml di liquido di scintillazione e mescolare. Misurare la radioattività in un contatore a scintillazione-β. </li></ol> 5. Riciclaggio i Beads <ol><li> Raccogliere le perline magnetiche usate in una provetta e centrifugare a 500 xg per 2 minuti o fino a un surnatante limpido. </li><li> Rimuovere il surnatante e risospendere le sfere in 6 ml 0.5 M NaOH. </li><li> Trasferire la sospensione multipltubi elettronici ed incubare in un bagno ad ultrasuoni per 5 minuti. </li><li> Utilizzare i magneti per raccogliere le biglie magnetiche e rimuovere il supernatante. </li><li> Aggiungere 1 ml di 2% SDS-soluzione a ciascuna provetta e utilizzando un vortice. Far bollire per 5 minuti. </li><li> Raccogliere le perline e rimuovere il supernatante. Risospendere le sfere in 1 ml di 0,2 M EDTA (pH = 7). </li><li> Incubare le provette in un bagno ad ultrasuoni per 5 minuti e ancora una volta, rimuovere il supernatante. </li><li> Aggiungere 1 ml di acqua a ciascuna provetta e le microsfere. Raccogliere le perline e rimuovere il supernatante. Ripetere questa fase di lavaggio due volte. </li><li> Rimuovere il surnatante e risospendere le sfere in 1 ml di 10 mM CoCl 2 soluzione. Mettere su un agitatore per 10 minuti. </li><li> Rimuovere il surnatante usando un magnete per raccogliere le perline e risospendere in 1 ml di Binding / tampone di lavaggio. </li><li> Rimuovere il surnatante e lavare le perline due volte con 1 ml di 20% (v / v) etanolo </li><li> Risospendere le sfere nel loro volume originale del 20% (v / v) ethanol ottenere perle rigenerate in una concentrazione di 40 mg / ml. </li></ol> 6. Interpretazione dei risultati <ol><li> Campione di controllo 1, in cui è stato aggiunto nessun virus radiomarcato, verrà usato per correggere la radioattività di fondo e per impostare il valore di radioattività 0%. Campione di controllo 2, che non contiene nanobody e dove di conseguenza tutti i virus radiomarcato rimane nel surnatante, viene impostato come valore radioattività 100%. </li><li> La percentuale di radioattività nel supernatante (= uno%) è una misura della precipitazione complessiva (= 100 - uno%) del virus dal radiomarcato nanobody. </li></ol> 7. Risultati rappresentativi Risultati rappresentativi per questo saggio di cattura affinità sono mostrate nelle Figure 2, 3 e 4. In figura 2, l&#39;affinità di PVSS38C, uno specifico per nanobody poliovirus, viene mostrato. L&#39;affinità di PVSS38C è stato testato per tre differenti antigeni di poliovIRU: Native-antigene (antigene N), Piscina riscaldata-antigene (antigene H) e la subunità 14S. N-antigene è il virus intatto, che è contagioso. Quando il virus viene riscaldata (20 minuti a 56 ° C) o attaccato ad una superficie solida (risultati non pubblicati), la conformazione dei cambiamenti del capside, risultando in un (parziale) perdita di RNA virale e la proteina del capside VP4, e vuoti capsidi sono formate che sono poi chiamati H-antigeni. Subunità 14S sono intermedi di montaggio. Questi antigeni sono riconosciuti da diversi set di anticorpi dopo immunizzazione 10 e quindi possiedono epitopi differenti e siti antigenici. Nella figura la percentuale di radioattività trovato nel supernatante è rappresentata come una funzione della concentrazione di nanobody. Si può vedere che la radioattività nel supernatante dei campioni contenenti radiomarcati subunità 14S diminuisce con concentrazioni crescenti del PVSS38C nanobody. Questo può essere tradotto come un aumento nella quantità di poliovirus con subunità 14Scollegato alla PVSS38C nanobody e, indirettamente, alle biglie magnetiche. Il titolo cattura (= concentrazione di nanobody necessario acquisire 50% di virus radiomarcato) può essere calcolato come graficamente 0,099 nM. No affinità PVSS38C per la N-antigenica e H-antigenica forma di poliovirus si osserva. Da questi dati viene interpretato PVSS38C che interagisce con un epitopo che è presente esclusivamente sulla subunità 14S e non sui due forme altre antigeniche di poliovirus. Per dimostrare che la perdita di radioattività dal supernatante è dovuta soltanto alla affinità specifica del nanobody verso i suoi antigeni, il saggio stesso è stata eseguita con NB1, uno nanobody generato contro il regolatore trascrizionale di lrpB sulfataricus Sulfolobus e noto per avere alcuna interazione con poliovirus qualsiasi antigene. Il risultato di questa prova viene mostrato nella Figura 3. Tutti virus radiomarcato rimane nei supernatanti mostrano che non vi è alcuna reattivitàNB1 contro le tre forme antigeniche di poliovirus. Riproducibilità dei risultati è stata testata ripetendo l&#39;esperimento per un totale di otto volte per un dato nanobody (cioè, PVSP29F) in giorni diversi e da persone diverse. I risultati sono mostrati nella Figura 4. Il valore medio di radioattività percentuale nel supernatante è stato calcolato per ciascuna concentrazione nanobody ed è rappresentato in corrispondenza della sua deviazione standard. <img alt="Figura 1." src="/files/ftp_upload/3937/3937fig1.jpg" /> Figura 1. Una panoramica del principio (A) e il metodo (B) del test. A. His-tag nanobodies che riconoscono specificamente un antigene poliovirus certo sarà in grado di interagire con le biglie magnetiche rivestite con cobalto e precipitare il virus marcato radioattivamente. Nanobodies B. His-tag vengono aggiunti al poliovirus radioattivo e incubate. Biglie magnetiche rivestite sono cobaltoaggiunto e separazione magnetica del antigene legato / legato viene eseguita. La radioattività del supernatante viene misurata e la quantità di antigene catturato può essere derivata. <img alt="Figura 2" src="/files/ftp_upload/3937/3937fig2.jpg" /> Figura 2. Magnetic perline cattura affinità di antigeni diversi da poliovirus nanobody PVSS38C. La percentuale di radioattività trovato nel supernatante è rappresentata come una funzione della concentrazione di PVSS38C. Per 14S una concentrazione-risposta può essere trovato, che mostra l&#39;interazione di PVSS38C con un epitopo esclusivamente presente 14S. Nessuna significativa interazione con N-, né H-antigene può essere rilevata, anche se non trascurabile-specifica cattura a concentrazioni molto elevate è notato. <img alt="Figura 3" src="/files/ftp_upload/3937/3937fig3.jpg" /> Figura 3. Magnetic perline cattura affinità di antigeni diversi da poliovirus nanobody NB1 </strong&gt;. La percentuale di radioattività trovato nel supernatante è rappresentata come una funzione della concentrazione di NB1. NB1 è noto per avere alcuna interazione con qualsiasi poliovirus-antigene o subunità. Poiché il 100% della radioattività si trova nel surnatante, nessun virus è stato non specificamente legato alla NB1. La perdita di radioattività in figura 2 è pertanto solo grazie alla specifica affinità del nanobody verso suoi antigeni. <img alt="Figura 4." src="/files/ftp_upload/3937/3937fig4.jpg" /> Figura 4. La riproducibilità del dosaggio. Il valore medio di radioattività percentuale nel supernatante è rappresentata come una funzione della concentrazione di nanobody. L&#39;esperimento è stato ripetuto otto volte in giorni diversi e da persone diverse. La deviazione standard è rappresentato come barre verticali.

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Nel protocollo, la radioattività dell&#39;antigene interagire con il nanobody è definita come la perdita di virus radiomarcato dal supernatante. Pertanto la quantità di radioattività precipitato (= 100 - uno%) (legato alle biglie magnetiche) può essere stimata in modo indiretto dalla radioattività nel surnatante (= uno%). D&#39;altra parte, è anche possibile misurare la radioattività della frazione legata precipitato di antigene in modo da eluendo degli immunocomplessi dalle perline magnetiche con imidazolo 500 ...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strongNome&gt; del reagente</strong</td&gt;<td<strong&gt; Azienda</strong</td&gt;<td<strong&gt; Numero di catalogo</strong</td&gt;<td<strong&gt; Commenti</strong</td&gt;</tr&gt;<tr&gt;<td&gt; Dynabeads His-Tag Isolamento e Pulldown</td&gt;<td&gt; Invitrogen</td&gt;<td&gt; 101.03D</td&gt;<td&gt; Particelle magnetiche</td&gt;</tr&gt;<tr&gt;<td&gt; Optiphase &#39;HiSafe&#39; 2</td&gt;<td&gt; Perkin Elmer</td&gt;<td&gt; 1200 - 436</td&gt;<td&gt; Scintillazione liquido</td&gt;</tr&gt;</tbody&gt;</table

a liquid phase affinity capture assay using magnetic beads to study protein-protein interaction: the poliovirus-nanobody example

In questo articolo, un semplice, quantitativa, liquido affinità saggio fase di cattura è presentato. A condizione che una proteina può essere etichettato e un&#39;altra proteina etichettatura, questo metodo può essere implementato per l&#39;istruttoria delle interazioni proteina-proteina. Esso si basa su un lato sul riconoscimento della proteina marcata con cobalto perline rivestite magnetiche e dall&#39;altro sulla interazione tra la proteina marcata e una seconda proteina specifica che viene etichettato. In primo luogo, le proteine ​​marcate e targhetta vengono miscelati e incubati a temperatura ambiente. Le biglie magnetiche, che riconoscono il comando, vengono aggiunti e la frazione legata di proteina marcata viene separata dalla frazione non legata utilizzando magneti. La quantità di proteina marcata che viene catturata può essere determinato in modo indiretto misurando il segnale della proteina marcata è rimasto nella frazione non legata. La fase di test descritto liquida affinità è estremamente utile quando le proteine ​​di conversione conformazionale sensibili sono assAyed. Lo sviluppo e l&#39;applicazione del test è stata dimostrata per l&#39;interazione tra poliovirus e poliovirus riconoscere nanobodies 1. Poiché poliovirus è sensibile alla conversione conformazionale 2 quando attaccato ad una superficie solida (risultati non pubblicati), l&#39;uso di ELISA è limitato e un sistema di fase liquida a base deve pertanto essere preferito. Un esempio di un sistema di fase liquida a base spesso utilizzati in polioresearch 3,4 è la proteina A micro-immunoprecipitazione test 5. Anche se questo test ha dimostrato la sua applicabilità, esso richiede una struttura-Fc, che è assente nelle nanobodies 6,7. Tuttavia, come un&#39;altra opportunità, questi interessanti e stabile singolo dominio anticorpi 8 può essere facilmente costruito con diversi tag. Il ampiamente utilizzato (His) 6-tag mostra affinità per ioni bivalenti come nichel o cobalto, che possono a loro volta essere facilmente rivestita su biglie magnetiche. Abbiamo quindi sviluppato questo quantitativo semplicecattura saggio di affinità a base di cobalto perline rivestite magnetiche. Poliovirus è stato marcato con 35 S per consentire l&#39;interazione con i nanobodies senza ostacoli e fare una rilevazione quantitativa fattibile. Il metodo è di facile esecuzione e può essere stabilita con un basso costo, che è ulteriormente supportata dalla possibilità di rigenerare efficacemente le biglie magnetiche.

Watch this Scientific Journal Video about A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example at JoVE.com

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

In questo articolo, un semplice, quantitativa, liquido affinità saggio fase di cattura è presentato. Si tratta di una tecnica affidabile basato sull&#39;interazione fra perline magnetiche e proteine ​​etichetta (ad esempio nanobodies) da un lato e l&#39;affinità tra la proteina marcata ed una seconda, proteina marcata (poliovirus esempio) dall&#39;altro.

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 ...

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Biology

11.8K Views.  Vrije Universiteit Brussel. In questo articolo, un semplice, quantitativa, liquido affinità saggio fase di cattura è presentato. Si tratta di una tecnica affidabile basato sull'interazione fra perline magnetiche e proteine ​​etichetta (ad esempio nanobodies) da un lato e l'affinità tra la proteina marcata ed una seconda, proteina marcata (poliovirus esempio) dall'altro.

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

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utilizzando il sistema GELFREE 8100 Frazionamento per peso molecolare basata su Frazionamento con recupero della fase liquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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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 ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Vescicole lipidiche-mediata Cromatografia di affinità mediante Magnetic separazione delle cellule attivate (LIMACS): un nuovo metodo per l&#39;analisi delle interazioni proteina-lipide

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

Conversione di un ELISA Capture ad un saggio Luminex xMAP utilizzando un anticorpo Multiplex metodo di screening

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

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. ...

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 ...

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of32 P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called &#8220;gel retardation&#8221; or &#8220;bandshift&#8221; assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

Electrophoretic Maiusc Mobility Assay (EMSA) per lo studio delle interazioni RNA-proteina: La IRE / IRP Esempio

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

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 ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.