Name: multimer-page: a method for capturing and resolving protein complexes in biological samples
Uploaded: 2017-05-05T14:00:00
Description: Watch this Scientific Journal Video about Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples at JoVE.com

Støttet af NIH / Nida DA034783. Vi takker Bryan A. Killinger til teknisk bistand med multimer-PAGE.

1. Vævspræparation <ol><li> Forbered 10 ml 4x BN-PAGE-prøvepuffer (200 mM Bis (2-hydroxyethyl) amino-tris (hydroxymethyl) methan (Bis-Tris), 200 mM NaCl, 40% vægt / volumen glycerol, 0,004% Ponceau S, pH 7,2 ). BEMÆRK: Denne stamopløsning kan fremstilles i forvejen og opbevares ved 4 ° C. </li><li> Fortynd 250 pi 4x prøvepuffer i 750 pi dH2O indeholdende 1 X kommerciel protease inhibitor cocktail. Vortex og chill på is. </li><li> Homogenisere 20 mg målvævet i 1 mL 1x iskold BN-PAGE-prøvep...

<ol>
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Protein-protein interaktioner er vigtige for enhver opgave levende ting udfører. På grund af dette, de er genstand for en intens undersøgelse og forskning. Multimer-PAGE er en ny fremgangsmåde til indfangning, separering og analyse af en lang række af proteinkomplekser. Vi har tidligere vist sin anvendelighed til at studere oligimerization af den sygdomsassocierede protein α-synuclein 11. Det er dog udvides til mange proteinkomplekser, som vist i figur 2. Sammenlignet med a...

<html> Normal cellulær funktion er afhængig af protein-protein interaktioner 1 , 2 . Som følge heraf er menneskelige sygdomme ofte præget af forstyrrelser i samlingen og opførelsen af ​​forskellige proteinkomplekser 3 . Evnen til at karakterisere sådanne interaktioner er derfor kritisk. Nuværende midler til at detektere disse interaktioner kræver rensning af målproteiner, ofte efterfulgt af nedtrængning af deres samspillende partnere. Konventionel oprensning udføres ved differentiel centrifugering, udfældning og / eller kromatografi 4 . Disse metoder er tidskrævende, skal ændres for hvert målprotein og resulterer ofte i lave udbytter. Moderne oprensningsmetoder involverer fusion af peptidmærker til målretning af proteiner efterfulgt af immunpræcipitation eller ekstraktion på søjler, der er fyldt med perler bundet til et fangsmolekyle 5 ,&quot;&gt; 6. Mens dette er strækbart for mange proteiner, kræver sekvens modifikation af målet, hvilket potentielt kan medføre konstruktioner, der afviger i affinitet for deres sædvanlige bindingspartnere. Den fine karakter af nogle protein-protein interaktioner betyder denne metode ikke kan anvendes til mange scenarier. Derudover pull-down assays anvendes til at kortlægge protein-protein-interaktioner kan ikke fange et nøjagtigt cellulær billede, på grund af begrænsede frihedsgrader og ikke-native niveauer af bait-proteinet. Ideelt set kunne proteinkomplekser påvises i deres native tilstande, uden behov for oprensning eller pull-down. Blå nativ polyacrylamidgelelektroforese (BN-PAGE) blev udviklet som en mindre-denaturerende alternativ til natriumdodecylsulfat-polyacrylamidgelelektroforese (SDS-PAGE), og giver mulighed for adskillelse af visse proteiner og komplekser fra biologiske prøver 7. Imidlertid proteiner i BN-PAGE adskille baseret på en large antal variabler, herunder størrelse, ladning, tredimensionelle struktur, og association med andre molekyler. Interaktionerne af disse faktorer resulterer ofte i co-separation af proteiner, aggregatdannelse og dårlig proteinbånd opløsning. Todimensional nativ polyacrylamidgelelektroforese løser nogle, men ikke alle, af disse problemer 8. At omgå de komplikationer forbundet med nativt separation, nogle forfattere anvender aminreaktive krydsbindingsreagenser, såsom dithiobis (succinimidylpropionat) (DSP), at indfange proteinkomplekser i vævslysater 4. Disse behandlede lysater kan derefter denatureres og adskilles ved SDS-PAGE, og samtidig bevare den native størrelse og makeup af proteinkomplekser. Men da krydsbindingsreagenser reagere ud fra nærhed af et molekyle til et andet, og proteiner i vævslysater har mange frihedsgrader og kan stokastisk interagere, specifik baggrund cross-linking kan være høj, især i koncentrerede prøver. Dette kan føre til svære at fortolke resultater. Her demonstrerer vi brugen af ​​en hybrid BN-PAGE / SDS-PAGE-metode, betegnet multimer-polyacrylamidgelelektroforese (multimer-PAGE) for at adskille og detektere proteinkonstruktioner i komplekse blandinger. I første omgang suspenderes cellelysat i polyacrylamidgel via BN-PAGE. Den lysatholdige gel omsættes derefter med tværbindingsreagens DSP. Pseudo-immobiliseret og lidt adskilt på gelen, er proteiner meget mindre tilbøjelige til at reagere uspecifikt, hvilket betyder, at baggrunds-tværbindingsreaktiviteten reduceres. Efter tværbinding udskilles gelbåndene og adskilles via SDS-PAGE. Den resulterende gel kan derefter analyseres på ethvert middel, der typisk er forbundet med polyacrylamidgelelektroforese. Denne metode muliggør adskillelse og påvisning af native proteinkomplekser i umodificeret vævsl lysat uden behov for yderligere rensning eller pull-down.

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			<td style="width:119px;">161-0148</td>
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			<td style="width:119px;">B9754</td>
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			<td style="width:119px;">A399-1</td>
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			<td style="width:119px;">24078</td>
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			<td style="width:119px;">22585</td>
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			<td>78430</td>
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			<td>For titration. Caustic.</td>
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			<td height="21" style="height:21px;width:336px;">N,N,N&#39;,N&#39;-tetramethylethylenediamine</td>
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			<td style="width:119px;">T9281</td>
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			<td style="width:119px;">16479</td>
			<td>Toxic.</td>
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			<td height="21" style="height:21px;width:336px;">NP40</td>
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			<td height="21" style="height:21px;width:336px;">Polysorbate 20 (tween-20)</td>
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			<td height="21" style="height:21px;width:336px;">Polyvinylidene fluoride transfer membranes</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">88518</td>
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			<td height="21" style="height:21px;width:336px;">Ponceau S</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">P3504</td>
			<td></td>
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			<td height="21" style="height:21px;width:336px;">Potassium chloride</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">P217-3</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Potassium phosphate monobasic</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">P9791</td>
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			<td height="21" style="height:21px;width:336px;">Protease inhibitor cocktail</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">88265</td>
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			<td height="21" style="height:21px;width:336px;">SDS solution (10% w/v)</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">161-0416</td>
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			<td height="21" style="height:21px;width:336px;">Sodium chloride</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP358-212</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Sodium dodecyl sulfate</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">L37771</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Sodium phosphate monobasic</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP329-1</td>
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			<td height="21" style="height:21px;width:336px;">Tricine</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">T0377</td>
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			<td height="21" style="height:21px;width:336px;">Tris base</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP152-500</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Tris-HCl (0.5 M buffer, pH 6.8)</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">161-0799</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Tris-HCl (1.5 M buffer, pH 8.8)</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">161-0798</td>
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			<td>Comments</td>
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			<td height="21" style="height:21px;width:336px;">GE Imagequant LAS 4000</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">28-9558-10</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">ImageJ software</td>
			<td style="width:195px;">NIH</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Synergy H1 microplate reader</td>
			<td style="width:195px;">BioTek</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Gel Former + Stand</td>
			<td style="width:195px;">Biorad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Microfuge 22R centrifuge</td>
			<td style="width:195px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">2 mL dounce homogenizer</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Vortex mixer</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Ultrasonic tissue homogenizer</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">FB120220</td>
			<td></td>
		</tr>
	</tbody>
</table>

multimer-page: a method for capturing and resolving protein complexes in biological samples

Der er mange veludviklede metoder til oprensning og studere enkelte proteiner og peptider. Men de fleste cellulære funktioner udføres af netværk af interagerende protein-komplekser, som ofte er vanskeligt at efterforske fordi deres binding er ikke-kovalent og let forstyrres af oprensningsteknikker. Dette arbejde beskriver en fremgangsmåde til stabilisering og adskillelse native proteinkomplekser fra umodificeret væv under anvendelse af todimensional polyacrylamidgelelektroforese. Tissue lysat påsat en ikke-denaturerende blå-nativ polyacrylamidgel, derefter en elektrisk strøm anvendes indtil proteinet migrerer en kort afstand ind i gelen. Gelen strimmel indeholdende det migrerede protein derpå udskåret og inkuberet med aminreaktive krydsbindingsreagens dithiobis (succinimidylpropionat), som covalent stabiliserer proteinkomplekser. Gelen strimmel indeholdende tværbundne komplekser derefter støbt i en natriumdodecylsulfat-polyacrylamidgel, og than komplekser adskilles fuldstændigt. Metoden bygger på teknikker og materialer er velkendte for de fleste molekylærbiologer, hvilket betyder at det er billigt og nemt at lære. Selv om det er begrænset i sin evne til i tilstrækkelig grad at adskille ekstremt store komplekser, og har ikke været universelt succes, metoden var i stand til at fange et bredt udvalg af velundersøgte komplekser, og er sandsynligvis gældende for mange systemer af interesse.

I denne demonstration eksperiment blev multimer-PAGE udført på hele rottehjerner lysat. De resulterende separerede proteiner blev blottet på polyvinyliden diflouride (PVDF) membraner, og derefter probet med antistoffer mod proteiner, der vides at danne komplekser. Figur 1 viser en validering af protokollen på to måder. Første viser vi, at de tværbundne proteiner er spaltelige ved tilsætning af et reduktionsmiddel, hvilket betyder de observerede højere molekylvæ...

Watch this Scientific Journal Video about Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples at JoVE.com

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

En fremgangsmåde til stabilisering og adskillelse native proteinkomplekser fra umodificeret væv lysat anvendelse af en amin-reaktivt protein tværbindingsmiddel koblet til en hidtil ukendt todimensional polyacrylamidgelelektroforese (PAGE) system er vist.

Multimer-PAGE: A Fremgangsmåde til registrering og Løsning proteinkomplekser i biologiske prøver

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Research

JoVE Journal

Biochemistry

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

Sheathless Capillary ElectrophoresisÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œMass Spectrometry for Metabolic Profiling of Biological Samples

Sheathless kapillarelektroforese-massespektrometri for Metabolic profilering biologiske prøver

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Fremgangsmåde til at identificere småmolekylære inhibitorer af protein-protein-vekselvirkning mellem HCN1 og TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Time-løst elektrosprayioniserings Brint-deuterium Exchange-massespektrometri til at studere proteiners struktur og Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Løsning affinitetsrensede proteinkomplekser ved Blå Native PAGE og protein Korrelation Profilering

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

Påvisning og visualisering af DNA-skader-inducerede proteinkomplekser i suspensionscellekulturer ved anvendelse af Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Horisontal gelelektroforese til forbedret påvisning af protein-RNA-komplekser

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

2 i 1: et-trins affinitet rensning for den parallelle analyse af Protein-Protein og Protein-metabolit komplekser

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

Brug af elektron paramagnetisk resonans i biologiske prøver ved omgivende temperatur og 77 K

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

Enkelt-trins rensning af makromolekylære komplekser ved hjælp af RNA knyttet til Biotin og et foto-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Bruger i Vitro fluorescens resonans energioverførsel at studere dynamikken i proteinkomplekser på en millisekund tidsskala

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...