Name: multimer-page: a method for capturing and resolving protein complexes in biological samples
Uploaded: 2017-05-05T14:00:00.000+00:00
Duration: 7 min 40 s
Description: Watch this Scientific Journal Video about Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples at JoVE.com

Unterstützt durch die NIH / NIDA DA034783. Wir danken Bryan A. Killinger für technische Unterstützung bei der Multimer-PAGE.

1. Gewebepräparation <ol><li> Herstellung von 10 ml der 4-fach BN-PAGE-Probenpuffer (200 mM Bis (2-hydroxyethyl) amino-tris (hydroxymethyl) methan (Bis-Tris), 200 mM NaCl, 40% w / v Glycerin, 0,004% Ponceau S, pH 7,2 ). HINWEIS: Diese Stammlösung im Voraus und bei 4 ° C durchgeführt werden kann. </li><li> Verdünnte 250 ul 4x Probenpuffer in 750 &amp; mgr; l dH 2 O , enthaltend 1x kommerziellen Protease - Inhibitor - Cocktail. Vortex und Chill auf Eis. </li><li> Homogenisiert 20 mg des Zielgewebes in den 1 ml 1x eiskalten BN-PAGE-Probenpuffern mit 30 Schlägen eines Dounce-Homogenisators sauber. HINWEIS: Für dieses Demonstrationsexperiment, das Zielgewebe ganze Rattenhirngewebe. Nach der Homogenisierung Proben werden mit einem milden Reinigungsmittel, wie beispielsweise 2% Digitonin zu solubilisieren und erlauben Elektrophorese von Membranproteinen behandelt. </li><li> Überträgt das Homogenisat in ein 1,5 ml Mikrozentrifugenröhrchen und zentrifugiert bei 14.000 xg für 30 min unlöslichen Zellinhalt zu pelletieren. after Zentrifugieren dekantiert den Überstand in ein sauberes Röhrchen auf Eis. </li><li> Folgen Sie den Anweisungen des Herstellers überstehende Flüssigkeit Proteinkonzentration unter Verwendung von Bicinchoninsäure (BCA) oder ähnlichen Proteinquantifizierung Assay zu messen. </li><li> Wenn das Homogenat Probe (n) Reinigungsmittel enthält, eine Menge von 5% Coomassie Blue G-250 in wässriger Lösung hinzu ausreichen, um die Lösung Homogenat Coomassie bis 0,25% zu bringen. </li></ol> 2. BN-PAGE <ol><li> Verdünne 25 ml 20x blau nativen (BN) Elektrophoresepuffer Lager (1,0 M Bis-Tris, 1,0 M Tricin, pH 6,8) in 475 ml dH 2 O 0,002% Coomassie - Blau , das 500 ml 1x Laufpuffer zu machen. <ol><li> Wenn Proben Reinigungsmittel enthalten, stellen stattdessen 250 ml 1x Laufpuffer, enthaltend 0,02% Coomassie und weitere 250 ml enthielt keine. </li></ol></li><li> Chill-Puffer (n) auf 4 ° C. </li><li> Reinigen und montieren ein Polyacrylamidgel Ausgießen Kassette nach Herstelleranweisungen. </li><li&gt; BN Polyacrylamidgel (3% T Stapelschicht, 6% T Auflösungsschicht) Gießen nach einem Standardrezept 7 und legen den gut Kamm. </li><li> Nachdem die Gele polymerisiert haben, spülen Sie die Elektrophoresevorrichtung gemäß Herstelleranweisungen , um die Kassette mit dH 2 O und zusammenzubauen. </li><li> Entfernen Sie die gut Kamm und füllen die Vertiefungen mit 1x BN-PAGE - Laufpuffer. Vermeiden die gesamte Innenkammer mit Puffer füllt , bis Proben geladen werden. <ol><li> Wenn Proben Waschmittel enthalten, füllen die Vertiefungen mit dem Puffer 0,02% Coomassie-Blau enthält. HINWEIS: Führen Sie die Schritte 2,7-2,9 bei 4 ° C. </li></ol></li><li> Verwendung von Gel-Ladespitzen, eine Pipette mit einem Volumen von Homogenisats, das 20 ug Protein in die gewünschten Vertiefungen. Pipette ein äquivalentes Volumen von 1x BN-PAGE-Probenpuffer, in einem nicht genutzten Vertiefungen. HINWEIS: Die Proteinkonzentration aus dem BCA-Test bestimmt das geeignete Volumen des Homogenats berechnen hinzuzufügenDie Brunnen. </li><li> Nachdem die Proben geladen sind, füllen Sie die innere Kammer mit 1x BN-PAGE-Laufpuffer. Seien Sie sicher, dass das Gel vollständig in Puffer untergetaucht ist. Als nächstes füllen Sie die Außenkammer mit 1x Laufpuffer auf das vom Hersteller angegebene Niveau. <ol><li> Wenn Proben Waschmittel enthalten, füllen Sie die innere Kammer mit dem 1x Puffer, der 0,02% Coomassie Blue enthält, und die äußere Kammer mit farbstofffreiem 1x Laufpuffer. </li></ol></li><li> Verbinden Sie die Elektroden mit der Stromversorgung und elektrophorieren Sie die Proteine ​​im Gel bei 150 V, bis das Farbstoffband ~ 2 cm in die Auflösungsschicht fortschreitet. Stoppen und trennen Sie die Stromversorgung. </li></ol> 3. Vernetzung HINWEIS: Die Schritte 3.1-3.6 bei 4 ° C durchführen. <ol><li> Demontieren Sie den Elektrophoreseapparat und trennen Sie die Glasscheiben der Gelkassette. Entfernen und entsorgen Sie die Stapelschicht. </li><li> Sorgfältig das Gel kurz unterhalb der Unterkante der Farbstofffront schneiden. NehmenPflege dieses Schnitt so glatt und gerade wie möglich zu machen, und dann die nicht verwendete Gelstück verwerfen. Dies wird einen ~ 2 cm breite, horizontale Streifen aus Polyacrylamidgel, läßt die alle das Protein in der Probe Homogenat enthalten. </li><li> Wegschneiden alle nicht verwendeten Teile entlang der Kanten des Gelstreifens. </li><li> Legen Sie das Band in 10 ml phosphatgepufferter Salzlösung (PBS) in einem kleinen Behälter, und sanft durch Nutation mischte für 30 Minuten äquilibrieren. </li><li> Nach der Äquilibrierung verwerfen und das PBS mit weiteren 10 mL ersetzen. Pipette 500 ul 25 mM DSP in Dimethylsulfoxid in das PBS gelöst und weiter wie oben (Schritt 3.4) für 30 min gemischt wurde. </li><li> Gießen Sie die DSP-Lösung ab. 10 ml 0,375 M Tris (hydroxymethyl) aminomethanhydrochlorid (Tris-HCl), pH 8,8, enthaltend 2% Natriumdodecylsulfat (SDS), um das nicht umgesetzte DSP quenchen. Weiter Nutation für 15 min. </li></ol> 4. SDS-PAGE <ol><li> Während Gelstreifen wird Abschrecken herzustellen SDS-PAGE Gellösungen nach Standardmethoden 9. Nicht Polymerisationsreagentien hinzuzufügen. </li><li> Nach dem Abschrecken auf Raumtemperatur zurückkehren die ΒΝ Gelstreifen, und der Streifen in eine neue Gelkassette gegossen. <ol><li> Um dies zu tun, nehmen Sie vorsichtig das Gel und legen Sie sie auf eine saubere Gelkassette Distanzplatte. </li><li> Orient der Streifen , so dass die Unterseite der Farbstofffront ist am nächsten an der Spitze der neuen Kassette (dh die Seite, die am weitesten in BN-PAGE gereist sollte auf der Oberseite der neuen Kassette am nächsten ist, der Gelstreifen aus seiner vorherigen umgedreht werden soll Orientierung). Siehe Abbildung 3. </li><li> Platzieren Sie den Streifen so , dass seine Oberkante liegt auch bei denen die Oberkante der Abdeckplatte wird ( das heißt, es wird an der Oberseite des neuen Gel sein). Sicherstellen, dass die Farbstofffront parallel zu den horizontalen Kanten der Glasplatte. </li><li> Schieben einer Seite des ausgeschnittenen Streifens gegen einen der Abstandshalterwände, so dass Raum für ter andere Seite für Gel gegossen werden und ein Protein-Standard oder Leiter geladen werden. </li><li> Wenn die Unterkante des Gelstreifens irgendwelche gezackten oder unebene Bereiche enthält, schneiden sie sorgfältig entfernt. Falls vorhanden, werden diese Blasen während trap Gelgießlösung. </li><li> Sobald die Gelstreifen korrekt positioniert ist, legte die Abdeckplatte über die Distanzplatte. Drücken Sie vorsichtig heraus drücken alle eingeschlossenen Luftblasen. </li><li> Weiterhin die gel-Gießvorrichtung nach Herstelleranleitung montieren. </li></ol></li><li> Hinzufügen Polymerisation Reagenzien zu dem Trenngel-Puffer und gießt es in die vorbereitete Gelkassette eine serologische Pipette. Füllen Sie das Gelkassette auf ~ 2 cm unterhalb der ausgeschnittenen BN-PAGE Gelstreifen, Raum für die Stapelung Schicht zu verlassen. </li><li> Füge 100 &amp; mgr; l Butanol über die oberen Rand des Gels gegossen, und ermöglicht es 30 Minuten zur Polymerisation des Auflösungsschicht. Gießen Sie das Butanol aus. </li><li> In Polymerisationsreagentien zur Stapel Gel - Lösung. Mit einem serologische Pipette, gieße die Stapelschicht alle verbleibenden Leerraum in der Gelkassette zu füllen. <ol><li> Neigen die Gelkassette als die Stapelschicht gegossen wird, so dass er den Raum unterhalb des Gelstreifens füllt, und Luftblasen werden nicht aufgefangen. </li><li> Da die Stapelung Gelpuffer den leeren Raum unter dem Gelstreifen füllt, kehrt allmählich die Gelkassette auf Augenhöhe. </li><li> neben dem exzidierten Gelstreifen mit dem Stapel Gelpuffer, bis es fast überläuft weiterhin den leeren Raum zu füllen. </li></ol></li><li> Ermöglicht die Stapelschicht für 30 Minuten zu polymerisieren. HINWEIS: Stellen 10x SDS-PAGE-Laufpuffer (250 mM Tris (hydroxymethyl) aminomethan (Tris), 1,9 M Glycin, 1% SDS), während das Gel polymerisiert. Dies kann auch im Vorfeld und bei Raumtemperatur gelagert gemacht werden. </li><li> Verdünne 50 mL 10X - SDS-PAGE - Laufpuffer Lager in 450 ml dH 2 O 1x Arbeitspuffer zu machen. </li><li> Nachdem die Stapelschicht polymerisiert ist, entfernen Sie die Gelkassette von strömendemVorrichtung, Spülen mit dH 2 O, und montiert die Elektrophoresevorrichtung nach Herstelleranweisungen. </li><li> Füllen der inneren Kammer vollständig mit 1x SDS-PAGE-Laufpuffer, dann die Außenkammer durch den Hersteller angegeben auf das Niveau füllen. </li><li> Laden den Raum neben dem exzidierten Gelstreifen mit einem Molekulargewicht Leiter oder geeigneten Proteinstandard. </li><li> Befestigen der Elektroden an die Stromversorgung, und Elektrophorese der Proben bei 120 V. Wenn der Farbstoff Coomassie das Gel abläuft, stoppen und die Stromversorgung trennen. </li><li> Analysieren des Gels unter Verwendung von Standardverfahren des Elektroblotting und Proteindetektion durch Antikörperbindung 10. </li></ol>

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Protein-Protein-Wechselwirkungen sind wichtig für jede Aufgabe Lebewesen auszuführen. Aus diesem Grunde sind sie Gegenstand intensiver Prüfung und Forschung. Multimer-PAGE ist ein neues Verfahren für die Erfassung, trennt, und eine Vielzahl von Proteinkomplexen zu analysieren. Wir haben bereits gezeigt , seine Anwendbarkeit oligimerization des krankheitsassoziierten Protein α-Synuclein 11 bis zu studieren. Jedoch ist es dehnbar zu vielen Proteinkomplexen, wie in Abbildung 2 ...

<html> Normale zelluläre Funktion ist abhängig von Protein-Protein - Wechselwirkungen 1, 2. Als Ergebnis werden menschliche Krankheiten , die oft durch Störungen in der Montage und Verhalten verschiedenen Proteinkomplexe 3 markiert. Die Fähigkeit, solche Interaktionen zu charakterisieren, ist daher von entscheidender Bedeutung. Aktuelle Mittel, um diese Wechselwirkungen zur Detektion erfordern Reinigung von Zielproteinen, die oft durch Pull-Down ihrer Interaktionspartner gefolgt. Herkömmliche Reinigung wird durch differentielle Zentrifugation, Präzipitation durchgeführt, und / oder Chromatographie 4. Diese Verfahren sind zeitaufwendig, muss für jedes Zielprotein verändert werden, und führen oft zu geringen Ausbeuten. Moderne Reinigungsverfahren umfassen die Fusion von Peptid - Tags an Zielproteine, durch Immunpräzipitation oder Extraktion auf Säulen , gefolgt mit Perlen zu einem Einfang - Moleküle gebunden 5 geladen wird ,„&gt; 6. Während dieses erweiterbaren viele Proteine ist, erfordert es Sequenzmodifikation des Ziels, möglicherweise in Konstrukten führt , die für ihre üblichen Bindungspartner in der Affinität unterscheiden. Die empfindliche Natur einiger Protein-Protein - Wechselwirkungen , bedeutet dieses Verfahren nicht anwendbar sein kann viele Szenarien. Außerdem ist das Pull-down-Assays verwendet, um Protein-Protein-Wechselwirkungen abbilden kann keine genaue zelluläre Bild erfassen, aufgrund eingeschränkter Freiheitsgrade und nicht-nativen Mengen des Köders Proteins. Idealerweise Proteinkomplexe könnten zur Reinigung oder Pull-Down ohne die Notwendigkeit, in ihren Heimatstaat nachgewiesen werden. Blau nativen Polyacrylamid - Gelelektrophorese (BN-PAGE) wurde als weniger denaturierende Alternative zu Natriumdodecylsulfat - Polyacrylamidgelelektrophorese (SDS-PAGE) entwickelt und erlaubt die Trennung von einigen Proteinen und Komplexen aus biologischen Proben 7. Eine getrennte Proteine ​​in BN-PAGE auf Basis eines LARge Anzahl von Variablen, einschließlich der Größe, Ladung, dreidimensionale Struktur, und die Assoziation mit anderen Molekülen. Die Wechselwirkungen dieser Faktoren führen häufig in Zusammenarbeit Trennung von Proteinen, die Aggregatbildung und schlechter Proteinbande Auflösung. Zweidimensionale nativer Polyacrylamid - Gelelektrophorese löst einige, aber nicht alle dieser Probleme 8. Um die Komplikationen zu umgehen mit nativer Trennung verbunden sind , verwenden einige Autoren aminreaktive Vernetzungsreagenzien, wie Dithiobis (succinimidylpropionat) (DSP), Proteinkomplexe in Gewebelysaten 4 zu erfassen. Diese behandelten Lysate können dann durch SDS-PAGE denaturiert und abgetrennt werden, während die native Größe und Zusammensetzung von Proteinkomplexen zu erhalten. Da jedoch die Vernetzungsreagenzien auf die Nähe von einem Molekül zum anderen auf Basis reagieren und Proteine ​​in Gewebe-Lysaten haben viele Freiheitsgrade und kann stochastisch interagieren, nicht-spezifischen Hintergrund Quer-Verknüpfung kann in konzentrierten Proben hoch, vor allem sein. Dies kann schwierig zu interpretierenden Ergebnissen führen zu. Hier stellen wir die Verwendung eines Hybrid-BN-PAGE / SDS-PAGE-Verfahren zeigen, bezeichnet als Multimer-Polyacrylamid-Gelelektrophorese (Multimer-PAGE), zu trennen, und das Protein Baugruppen in komplexen Mischungen zu erfassen. Anfänglich wird Zelllysat in Polyacrylamidgel über BN-PAGE suspendiert. Das Lysat enthaltende Gel wird dann mit dem Vernetzungsreagenz DSP. Pseudo-immobilisierte und leicht auf dem Gel aufgetrennt, Proteine ​​sind viel weniger wahrscheinlich unspezifisch zu reagieren, was bedeutet, Hintergrund Vernetzers Reaktivität verringert wird. Nach der Vernetzung werden die Gel-Banden ausgeschnitten und mittels SDS-PAGE getrennt. Das resultierende Gel kann dann durch ein beliebiges Mittel typischerweise mit Polyacrylamidgelelektrophorese assoziiert analysiert werden. Dieses Verfahren ermöglicht die Trennung und den Nachweis von nativen Proteinkomplexen in unmodifizierter Gewebelysat, ohne die Notwendigkeit für eine zusätzliche Reinigung oder Pull-Down.

<table><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;epsilon;-Aminocaproic acid</td><td>Sigma</td><td>A2504</td><td></td></tr><tr><td>Acrylamide</td><td>Acros Organics</td><td>164855000</td><td>Toxic.</td></tr><tr><td>Acrylamide/bisacrilamide 37.5:1 (40%T stock solution)</td><td>BioRad</td><td>161-0148</td><td>Toxic.</td></tr><tr><td>Ammonium persulfate</td><td>Sigma</td><td>A3678</td><td></td></tr><tr><td>Anti rabbit IgG-HRP from goat</td><td>Santa Cruz Biotechnology</td><td>sc-2004</td><td></td></tr><tr><td>Bicinchoninic acid assay kit</td><td>Thermofisher</td><td>23225</td><td></td></tr><tr><td>Bis-tris</td><td>Sigma</td><td>B9754</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A9647</td><td></td></tr><tr><td>Butanol</td><td>Fisher Scientific</td><td>A399-1</td><td></td></tr><tr><td>Chemiluminescence substrate kit</td><td>ThermoFisher</td><td>24078</td><td></td></tr><tr><td>Coomassie blue G-250</td><td>Sigma-Aldrich</td><td>B0770</td><td></td></tr><tr><td>Digitonin</td><td>Sigma</td><td>D141</td><td>Toxic.</td></tr><tr><td>Dimethyl sulfoxide</td><td>Fisher Scientific</td><td>D128-1</td><td></td></tr><tr><td>Dithiobis(succinimidylpropionate)</td><td>Thermo Scientific</td><td>22585</td><td></td></tr><tr><td>Dry nonfat milk</td><td>LabScientific</td><td>M0841</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G9012</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP381-5</td><td></td></tr><tr><td>Halt Protease Inhibitor Cocktail</td><td>Thermofisher</td><td>78430</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific</td><td>A144SI-212</td><td>For titration. Caustic.</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A412-4</td><td>For PVDF membrane activation. Toxic.</td></tr><tr><td>Monoclonal anti &amp;alpha;-synuclein IgG from rabbit</td><td>Santa Cruz Biotechnology</td><td>sc-7011-R</td><td></td></tr><tr><td>N,N,N&#039;,N&#039;-tetramethylethylenediamine</td><td>Sigma</td><td>T9281</td><td></td></tr><tr><td>N,N&#039;-methylenebisacrylamide</td><td>Acros Organics</td><td>16479</td><td>Toxic.</td></tr><tr><td>NP40</td><td>Boston Bioproducts</td><td>P-872</td><td></td></tr><tr><td>Polysorbate 20 (tween-20)</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>Polyvinylidene fluoride transfer membranes</td><td>Thermo Scientific</td><td>88518</td><td></td></tr><tr><td>Ponceau S</td><td>Sigma</td><td>P3504</td><td></td></tr><tr><td>Potassium chloride</td><td>Fisher Scientific</td><td>P217-3</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P9791</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Thermo Scientific</td><td>88265</td><td></td></tr><tr><td>SDS solution (10% w/v)</td><td>BioRad</td><td>161-0416</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma</td><td>L37771</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Fisher Scientific</td><td>BP329-1</td><td></td></tr><tr><td>Tricine</td><td>Sigma</td><td>T0377</td><td></td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152-500</td><td></td></tr><tr><td>Tris-HCl (0.5 M buffer, pH 6.8)</td><td>BioRad</td><td>161-0799</td><td></td></tr><tr><td>Tris-HCl (1.5 M buffer, pH 8.8)</td><td>BioRad</td><td>161-0798</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GE Imagequant LAS 4000</td><td>GE Healthcare</td><td>28-9558-10</td><td></td></tr><tr><td>ImageJ software</td><td>NIH</td><td></td><td></td></tr><tr><td>Synergy H1 microplate reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>Gel Former + Stand</td><td>Biorad</td><td></td><td></td></tr><tr><td>Microfuge 22R centrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>2 mL dounce homogenizer</td><td></td><td></td><td></td></tr><tr><td>Vortex mixer</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Ultrasonic tissue homogenizer</td><td>Fisher Scientific</td><td>FB120220</td><td></td></tr></tbody></table>

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

Es gibt viele gut entwickelte Methoden zur Reinigung und einzelne Proteine ​​und Peptide zu studieren. Allerdings sind die meisten zellulären Funktionen von Netzwerken interagierender Proteinkomplexen durchgeführt, die oft schwierig zu untersuchen, da ihre Bindung nicht-kovalent und leicht durch Reinigungstechniken gestört. Diese Arbeit beschreibt ein Verfahren zur Stabilisierung und Trenn native Protein-Komplexe aus nicht-veränderten Geweben zweidimensionale Polyacrylamid-Gelelektrophorese unter Verwendung von. Tissue-Lysat wird auf eine nicht-denaturierenden blau-native Polyacrylamidgel geladen, dann wird ein elektrischer Strom angelegt wird, bis das Protein, das eine kurze Strecke in das Gel wandert. Die Gelstreifen die migrierten Protein enthält, wird dann mit den aminreaktiven Vernetzungsreagenz Dithiobis (succinimidylpropionat), das kovalent Protein ausgeschnitten und inkubiert Komplexe stabilisiert. Die Gelstreifen vernetzten Komplexe enthalten, werden dann ein Natriumdodecylsulfat-Polyacrylamid-Gel eingegossen, und tEr komplexe sind vollständig getrennt. Die Methode beruht auf Techniken und Materialien, die den meisten Molekularbiologen vertraut sind, was bedeutet, dass es kostengünstig und leicht zu erlernen ist. Während es in seiner Fähigkeit, äußerst große Komplexe adäquat zu trennen, begrenzt ist und nicht allgemein erfolgreich war, war die Methode in der Lage, eine Vielzahl von gut untersuchten Komplexen zu erfassen und ist wahrscheinlich auf viele interessierende Systeme anwendbar.

In dieser Demonstration Experiment wurde Multimer-PAGE auf ganzem Rattenhirn-Lysat durchgeführt. Die resultierenden getrennten Proteine ​​wurden auf Polyvinylidendifluorid (PVDF) Membranen geblottet und dann mit Antikörpern gegen Proteine ​​untersucht, die Komplexe sind dafür bekannt, zu bilden. Abbildung 1 zeigt eine Validierung des Protokolls durch zwei Mittel. Erstens, zeigen wir , dass die vernetzten Proteine durch Zugabe eines Reduktionsmittels abspaltbar...

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

Verfahren zur Stabilisierung und Trenn nativen Proteinkomplexe aus unmodifizierten Gewebelysat gekoppelt, um ein Amin-reaktives Protein Vernetzers unter Verwendung von auf eine neuartige zweidimensionale Polyacrylamid-Gelelektrophorese (PAGE) -System dargestellt.

Multimer-PAGE: Ein Verfahren zum Erfassen und Auflösen von Proteinkomplexen in biologischen Proben

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

multimer-page-method-for-capturing-resolving-protein-complexes

Research

JoVE Journal

Biochemistry

11.3K Aufrufe.  Michigan State University.  Verfahren zur Stabilisierung und Trenn nativen Proteinkomplexe aus unmodifizierten Gewebelysat gekoppelt, um ein Amin-reaktives Protein Vernetzers unter Verwendung von auf eine neuartige zweidimensionale Polyacrylamid-Gelelektrophorese (PAGE) -System dargestellt. 

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

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Protein Complex Affinity Capture von Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Forschung

Biochemie

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

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Auflösen von Affinity Purified Protein-Komplexen von Blue native PAGE und Protein Correlation Profilieren

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

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

Analyse dynamischer Proteinkomplexe montiert auf und befreit Biolayer Interferometrie Biosensor mit Massenspektrometrie und Elektronenmikroskopie

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Etikettenfreier Immunpräzipitations-Massenspektrometrie-Workflow für großangelegtes Nuklear-Interactome-Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Isolierung von Labile Multi-Protein-Komplexe durch In vivo Kontrollierte Cellular Cross-Linking und Immuno-magnetische Affinitätschromatographie

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Biologie

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Analyse großer Proteinkomplexe von Structural-Massenspektrometrie

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identifizierung von Protein-Komplexen in<em&gt; Escherichia coli</em&gt; Mit Sequential Peptide Affinity Purification in Kombination mit Tandem-Massenspektrometrie

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Die MultiBac Protein Complex Produktion Platform am EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion 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.

Die Entdeckung und Charakterisierung von Protein-Wechselwirkungen Protein-Funktion Mit HaloTag Technologie

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