Die Autoren danken Dr. Jim Lah und Allan Levey, Emory Universitätsklinik für Neurologie, für die hilfreichen Kommentare und Anregungen. Diese Arbeit wurde teilweise finanziert durch die Beschleunigung der Medizin Partnerschaft Grant (U01AG046161-02), die Emory Alzheimer Disease Research Center (P50AG025688) und eine National Institute on Aging gewähren (R01AG053960-01) N.T.S. Diese Forschung wurde auch teilweise durch die Neuropathologie Kern von Emory Neurowissenschaften NINDS Core Facilities Grant, P30NS055077 unterstützt.

Ethik-Erklärung: alle Hirngewebe stammen aus der Emory Alzheimer &#39; s Disease Research Center (ADRC) Brain Bank. Post-mortem Gewebe unter richtigen institutionellen Review Board (IRB) Protokolle erworben wurden.
 1. Homogenisierung und Fraktionierung <ol><li>Gewebe Auswahl Hinweis: Frozen Post-mortem frontalen Kortex Gewebe aus gesunden Kontrolle (Ctl) und pathologisch bestätigten AD-Fälle wurden ausgewählt von der Emory ADRC Gehirn Bank (n = 2). Post-Mortem neuropathologische Auswertung der Amyloid-Plaques Verteilung wurde entsprechend des Konsortiums, eine Registrierung für Alzheimer zu etablieren durchgeführt &#39; s Krankheit semi-quantitativen Kriterien 24, scoring, obwohl neurofibrillären Gewirr Pathologie wurde gemäß der Braak-staging-System bewertet. 16 <ol><li>erhalten Post-mortem Hirngewebe von gesunden Kontrolle (Ctl) und pathologisch eingefroren bestätigt AD-Fälle. Verwendung von Containern von Trockeneis, Zange und einer Rasierklinge, Verbrauchsteuern ~ 250 mg Teile der grauen Substanz aus jede Gewebeprobe auf tarierten Einweg wiegen Boote, kümmert sich um Auftauen des Gewebes zu verhindern. Nehmen Sie das Gewicht jedes Stückes, homogenisiert werden.</li>
		<li>Verbrauchsteuern ~ 250 mg der grauen Zellen mit Zange und gestochen von visuell inspizieren, um die Schnittstelle zwischen der äußeren graue Substanz und inneren weißen Substanz zu finden. Vermeiden Sie weiße Substanz und was noch wichtiger ist, alle großen Blutgefäße oder blutigen Regionen, Hirnhäute oder Arachnoidea Mater. Halten Sie das Gewebe eingefroren während des Schneidprozesses indem arbeiten schnell und häufig mit einem Gewicht von Boot mit Hirngewebe in Polystyrol-Behälter mit trocken Ice</li>
		<li>Datensatz das exakte Gewicht der grauen Substanz, die aus jedem gefrorenen Stück mit Hilfe einer Analysenwaage herausgeschnitten. Berechnen Sie das Volumen des wenig Salz (LS) Puffer für Dounce Homogenisierung (5 mL/g oder 20 % w/V) benötigt (siehe Tabelle 1).</li>
		<li>Vorbereitung 10 mL LS Puffer mit 1 x Protease und Phosphatase-Inhibitor cocktail und chill auf Eis.</li>
		<li>Entfernen Sie wog Gewebe und mit einem Gewicht von Boot aus Trockeneis und Würfel in etwa 2 x 2 mm Stücke wie es taut. Transfer zu einem 2 mL vorgekühlt Dounce Homogenisator Schlauch auf Ice</li>
		<li>5 mL/g (20 % w/V) der eiskalten wenig Salz (LS) Puffer mit 1 X Protease und Phosphatase-Inhibitor cocktail hinzufügen.</li>
		<li>Homogenize das Hirngewebe auf Eis mit etwa 10 Schläge von hoher Bodenfreiheit Stößel A und 15 Hübe der niedrige Clearance Stößel B.</li>
		<li>Alle Homogenat auf ein 2 mL Polypropylen-Rohr mit einem Glas Pasteurpipette übertragen. Beschriften der Röhrchen mit &#8220; TH &#8221; (insgesamt Homogenat), das Gewebe Fall Nummer und homogenisierte Volumen. aliquoten 0,8 mL in einem beschrifteten 1,5 mL-Tube. Jede überschüssige Homogenat kann ähnlich sein, regelmÄÑig und gespeicherte bei-80 ° c</li>
		<li>Zu je 0,8 mL Aliquoten, bzw. 100 µL 5 M NaCl und 10 % (w/V) Sarkosyl zu Konzentrationen von 0,5 M und 1 % w/V, hinzufügen. Mischen Sie Rohre gut durch Umkehrung und inkubieren Sie auf Eis für 15 min. Label Rohre mit &#34; TH-S &#34; (insgesamt Homogenat-Sarkosyl) darauf hin, dass die Homogenates in Sarkosyl-Puffer (Tabelle 1).</li>
		<li>Beschallen jedes Rohr für drei 5 s-Pulse bei 30 % Amplitude auf Eis (maximale Intensität = 40 %) mit einer Sonde Microtip.</li> <li>bestimmen die Proteinkonzentrationen von der Homogenates mit der Bicinchoninic Säure (BCA) assay 25 Methode. 
		Hinweis: Die durchschnittliche Proteinkonzentration von TH-S Homogenates in 5 mL LS pro Gramm Gewebe vorbereitet ist 15-20 mg/mL. Die Homogenates fraktioniert sofort oder bis zur Verwendung bei-80 ° C gelagert werden kann.</li>
		<li>Verdünnen TH-S Homogenates bis 10 mg/mL mit eiskalten Sark Puffer (Tabelle 1) mit 1 x Phosphatase, Protease-Inhibitoren.</li>
		<li>Transfer 5 mg Protein (0,5 mL) von jeder TH-S Homogenat in 500 µL Polycarbonat Ultrazentrifugen Tuben und paar-Balance mit Sark-Puffer. Laden Sie Rohre in einem vorgekühlt Rotor und Ultrazentrifugen 180.000 x g für 30 min bei 4 ° c Übertragung Sarkosyl lösliche Überstände (S1) auf 1,5 mL Röhrchen und Lagern bei-80 ° c 
		Hinweis: (Optional-Waschanlagen) fügen Sie 200 µL Sark-Puffer, der Ultrazentrifuge Rohre mit Waschmittel-unlösliche Fraktionen (P1) und verdrängen die Pellets (P1) von der Unterseite der Ultrazentrifuge Rohre mit einer Pipettenspitze 200 µL.</li>
		<li>Kurz Puls-Spin die invertierten Rohre für 2-3 s (&#8804; 2.500 X g) auf einem Microcentrifuge der Pellets (P1) und Puffer auf die 1,5 mL Röhrchen unten übertragen. Aufschwemmen der unlöslichen Pellets (P1) im Sark-Puffer durch Pipettieren rauf und runter um sicherzustellen das Pellet wird gestört,.</li>
		<li>Paar-Balance und Zentrifuge bei 180.000 x g für eine zusätzliche 30 min bei 4 ° c</li>
		<li>Verwerfen den optionalen waschen überstand (S2) und inkubieren Sie die Sarkosyl-unlösliche Pellets (P2) in 50-75 µL Harnstoff Puffer (Tabelle 1) mit 1 x PIC (Protease und Phosphatase-Inhibitor cocktail) für 30 min bei Raumtemperatur zu solubilisieren die Pellet. 
		Hinweis: Warme Harnstoff Puffer auf Raumtemperatur vor Gebrauch SDS Niederschlag zu vermeiden. Für Waschmittel-Sensitive Anwendungen weglassen SDS aus Harnstoff Puffer und in niedrigen Salz Puffer betrachten waschen das unlösliche Pellet (P2), bevor in Harnstoff Puffer resuspending.</li>
		<li>Übertragen die resuspendierte Pellets (P2) auf 0,5 mL Röhrchen und verwenden Sie kurze (1 s) Microtip Beschallung bei 20 % Amplitude (maximale Intensität = 40 %), voll die Pellets solubilisieren.</li>
		<li>Bestimmen die Proteinkonzentrationen von Sarkosyl löslich (S1) und unlösliche (P2) Brüche mit der BCA assay Methode. Diese Fraktionen sofort verwenden oder bei-80 ° C bis zur Verwendung aufbewahren.</li>
	</ol></li></ol> 2. Immunoblotting <ol><li>Western Blot Hinweis: Western blotting nach zuvor gemeldeten Verfahren mit leichten Modifikationen durchgeführt wurde. 5 , 10 <ol><li>bereiten 40 µg Proben von insgesamt Homogenates (TH-S), Sark-löslich (S1) und Sark-unlösliche (P2) Brüche in 1 X Laemmli SDS-Page-Probenpuffer mit 5 mM DÄMMUND pH 7. Inkubieren Sie Proben bei 95 ° C für 5 min.</li>
		<li>Last Proben auf ein Fertigteil 10-Brunnen 4-12 % Bis-Tris SDS-PAGE gel. Bei 80 V für die ersten Zentimeter (10 min) und 120 V für den Rest (60 min) oder bis die Tracking-Farbstoff den Boden des Gels erreicht electrophorese.</li>
		<li>Verwenden Sie frischen Rasierklinge und Gel Messer auf Split-die vorgegossenen Gel-Kassette offen. Sanft die Kämme und ganz unten das Gel mit einer frischen Rasierklinge wegschneiden.</li>
		<li>Aufschwemmen der unlöslichen Pellets (P1) in 200 µl Sark-Puffer mit 1 X PIC durch Pipettieren rauf und runter.</li>
		<li>Übertragung an eine 0,2 µm Nitrozellulose-Membran mit einer trockenen Übertragungsmethode auf ein Blot Maschine (siehe der Materialtabelle).</li>
		<li>Blockieren die Membran in blockierende Puffer (BB) ohne Tween 20 für 30 min, gefolgt mit BB mit 0,05 % Tween 20 (BB/T) für 30 min. Nach der Blockierung, spülen Sie die Membran in TBS/T (0,1 % Tween 20) für 5 min um überschüssige blockierende Puffer entfernen.</li>
		<li>Bereiten 1:1,000 Verdünnungen der primären Antikörper pT231, Tau-2 (Pan Tau), AT8 und EEA1 in BB/T (0,05 % Tween 20).</li>
		<li>Brüten die Membranen in Primärantikörper Lösung über Nacht bei 4 ° C mit kreisförmigen Bewegung. Spülen Sie die Membran in TBS/T für 3 x 15 min.</li>
		<li>Sonde die Membran mit einer Verdünnung 1: 20.000 Eindringmittel beschriftet in der Nähe von IR Sekundärantikörper in BB/T für 60 min bei Raumtemperatur in Dunkelheit auf Shaker. Spülen Sie die Membran für 3 x 10 min in TBS/T und 2 x 5 min in El</li>
		<li>Scannen die Membran, die über eine Infrarot imaging-System bei der entsprechenden Erregung Wellenlänge (z.B. 700 nm (Rot-Kanal) für Infrarot-Farbstoff 680 Ziege Anti-Maus IgG (H + L) sekundär- und 800 nm (Grün-Kanal) für Infrarot-Farbstoff 790 Esel Anti-Kaninchen IgG (H + L (sekundäre)).</li>
		<li>Verwenden die Software zur Abbilderstellung Signalintensitäten zu quantifizieren und Densitometrie Messungen gemäß den Hersteller &#39; Anweisungen, Auto-Hintergrund zu gewährleisten wird voraussichtlich den gesamten Hintergrund durchschnittliche.</li>
	</ol>
	</li>
</ol>

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Hier wir vorstellen und diskutieren eine abgekürzte Einzelschritt Waschmittel-Fraktionierung-Protokoll, die für eine Vielzahl von experimentellen Anwendungen von Massenspektrometrie-basierte Proteomics Analysen bis hin zu funktionellen Protein misfolding assays und westliche Beflecken. 5 , 6 , 7 , 10 dieser Methodik ist vielleicht die meisten wirksam, wenn Sie zur Untersuchung neurodegenerati...

Neurodegenerative Erkrankungen wie Alzheimer-Krankheit (AD), Parkinson-Krankheit (PD), Chorea Huntington (HD), Amyotrophe Lateralsklerose (ALS) und die eng verwandten Prion-Krankheiten sind gekennzeichnet durch die allmähliche Anhäufung von proteinopathies Waschmittel-unlösliche Aggregate im Gehirn mit begleitenden kognitiven ablehnen. 1 , 2 diese gemeinsame pathologische Funktion wird gedacht, um eine zentrale Rolle in der Ätiologie und Pathophysiologie dieser neurodegenerativen Krankheiten. 2 diese Aggregate bestehen typischerweise aus Polymeren Amyloid Fasern, die zusammengesetzt sind, sich wiederholender Einheiten der fehlgefaltete Protein ausstellenden Kreuz β-Struktur. 1 , 2 , 3 , 4 biochemisch, Amyloid Aggregate sind sehr widerstandsfähig gegen chemische oder thermische Denaturierung und Solubilisierung,3 stellt besondere Herausforderungen an ihre Reinigung, Analyse und über traditionelle biochemischen Techniken zu studieren. 2 , 5 , 6 , 7 , 8 , 9 , 10 , 11 es überrascht nicht, wurde die Waschmittel-unlösliche Proteinfraktion viel Forschungsschwerpunkt in der Pathophysiologie der neurodegenerativen Erkrankungen mit der Anhäufung von fehlgefaltete Proteine. 6 , 12 , 13 , 14
Biochemische Fraktionierung Techniken wurden oft genutzt, um die Reinigungsmittel-unlösliche Fraktion aus Post-mortem Gehirn Homogenates bereichern. 6 , 12 , 13 , 14 eine der am häufigsten verwendeten Methoden beinhaltet die sequentielle Extraktion von Gewebe Homogenates mit Puffern und Reinigungsmittel erhöhen Stringenz, gefolgt von Ultrazentrifugation, die löslichen und unlöslichen Anteile zu partitionieren. Eine häufig verwendete sequentielle Fraktionierung Protokoll6,14 beinhaltet die Homogenisierung der gefrorene Gewebeproben in einem Waschmittel-freie wenig Salz (LS)-Puffer und die daraus resultierende unlösliche Pellets werden dann nacheinander mit extrahiert Puffer mit hohen Salz, nichtionischen Detergentien, hohen Saccharose, wie Ionischen Detergentien und schließlich Chaotropes Harnstoff. 6 , 14 eine offensichtliche Nachteil solch eine sequentielle Fraktionierung Protokolle ist die beträchtliche Zeit und Arbeit Engagement erforderlich, um sie abzuschließen. Einschließlich der Homogenisierung und Ultrazentrifugation, eine typische 5-stufige Fraktionierung Protokoll dauert mehrere Stunden oder sogar Tage dauern. 4 , 6 , 7 , 10 , 15 , 16 , 17 , 18 darüber hinaus möglichst viele pathologische Proteinaggregaten unlöslich in allen außer den härtesten Bedingungen19,20 bleiben die meisten der erzeugten Brüche sind nur von begrenztem Wert. Somit sind die weniger strengen Fraktionierung Schritte nutzen hohe Salzkonzentrationen und nichtionische Detergentien weitgehend überflüssig.
Frühere Studien haben gezeigt, dass die ionische Waschmittel N-Natriumlaurylsulfat-Sarkosin (Sarkosyl) ein ausgezeichneter Kandidat für einen vereinfachten einstufigen Waschmittel Fraktionierung Protokoll ist. 5 , 6 , 12 , 13 , 14 , 21 , 22 , 23 als denaturierenden Waschmittel, Sarkosyl ist streng genug, um die überwiegende Mehrheit der nativ gefaltete Proteine im Gehirn anderweitig ohne Gefäss-fehlgefaltete Protein-Aggregate bestehend aus Beta-Amyloid (Aβ),6,11 phosphorylierten Tau (pTau),6 TAR DNA-bindendes Protein 43 (TDP-43),14 Alpha-Synuclein,12,13 Scrapie,23 oder U1 small nuclear Ribonucleoproteins (U1 SnRNPs) wie U1 - 70 K. 5 , 21 , 22 Da Sarkosyl weniger streng als die allgegenwärtigen anionische Detergenzien Dodecyl Natriumsulfat (SDS) ist, bewahrt es weniger robuste Oligomere Formen von fehlgefaltete Protein-Aggregate, die SDS-Behandlung nicht standhalten können. 9
Zuvor beschrieben wir eine abgekürzte Waschmittel-Fraktionierung-Protokoll, das vergleichbar mit aufwendiger sequentielle Fraktionierung Methoden Ergebnisse erzielt. 5 durch die weniger strenge Fraktionierung Schritte auslassen, konnten wir ein facile Einzelschritt Fraktionierung-Protokoll für die Bereicherung der Waschmittel-unlösliche Aggregate von Post-mortem Gehirn zu entwickeln. 5 dieses ausführliche Protokoll beschriebenen eignet sich gut für eine Vielzahl von experimentellen Anwendungen reichen von Western blotting und Massenspektrometrie-basierte Proteomics, funktionsfähiges Protein misfolding und Aggregation seeding assays. 5 , 6 , 21

<table>
	<tbody>
		<tr>
			<td>Protease and phosphatase inhibitor cocktail, EDTA-free (100X)</td>
			<td>Thermo Fisher</td>
			<td>78441</td>
			<td>protease &#38; phosphatase inhibitor cocktail</td>
		</tr>
		<tr>
			<td>Sonic Dismembrator System (ultrasonicator)</td>
			<td>Fisher Scientific</td>
			<td>FB505110</td>
			<td>microtip ultrasonicator</td>
		</tr>
		<tr>
			<td>Optimax TLX Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>361545</td>
			<td>refrigerated ultracentrifuge</td>
		</tr>
		<tr>
			<td>TLA120.1 rotor</td>
			<td>Beckman Coulter</td>
			<td>362224</td>
			<td>ultracentrifuge rotor</td>
		</tr>
		<tr>
			<td>500 ul (8x34mm) polycarbonate tubes, thickwall</td>
			<td>Beckman Coulter</td>
			<td>343776</td>
			<td>ultracentrifuge tubes for TLA120.1 rotor</td>
		</tr>
		<tr>
			<td>4X SDS sample buffer</td>
			<td>Home-made</td>
			<td>N/A</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>TCEP solution, neutral pH</td>
			<td>Thermo Fisher</td>
			<td>77720</td>
			<td>reducing agent</td>
		</tr>
		<tr>
			<td>(TBS) blocking buffer</td>
			<td>Thermo Fisher</td>
			<td>37542</td>
			<td>blocking buffer</td>
		</tr>
		<tr>
			<td>(TBS) blocking buffer + 0.05% Tween 20</td>
			<td>Thermo Fisher</td>
			<td>37543</td>
			<td>blocking buffer and antibody diluent</td>
		</tr>
		<tr>
			<td>4-12% Bolt Bis-Tris Plus gels, 10-well</td>
			<td>Thermo Fisher</td>
			<td>NW04120BOX</td>
			<td>precast SDS-PAGE gels</td>
		</tr>
		<tr>
			<td>MES SDS Running Buffer (20X)</td>
			<td>Thermo Fisher</td>
			<td>B0002</td>
			<td>SDS-PAGE running buffer</td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine sodium salt (sarkosyl)</td>
			<td>Sigma Aldrich</td>
			<td>L5777-50G</td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>1.5 ml polypropylene Pellet Pestle</td>
			<td>Kimble Chase</td>
			<td>749521-1500</td>
			<td>homogenization tool</td>
		</tr>
		<tr>
			<td>cordless motor for Pellet Pestle</td>
			<td>Kimble Chase</td>
			<td>749540-0000</td>
			<td>homogenization tool</td>
		</tr>
		<tr>
			<td>Anti-Tau-2 (pan tau) antibody</td>
			<td>Chemicon</td>
			<td>MAB375</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Anti-phospho-threonine 231 Tau antibody</td>
			<td>Millipore</td>
			<td>MAB5450</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Anti-phospho-seroine 202 and threonine 205 Tau antibody (AT8)</td>
			<td>Thermo Fisher</td>
			<td>MN1020</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Anti-early endosome antigen 1 (EEA1) antibody</td>
			<td>abcam</td>
			<td>ab2900</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Alexa Fluor 680 goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>Thermo Fisher</td>
			<td>A21058</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Alexa Fluor 790 donkey anti-rabbit IgG (H+L) secondary antibody</td>
			<td>Thermo Fisher</td>
			<td>A11374</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>iBlot2 Dry Blotting System</td>
			<td>Thermo Fisher</td>
			<td>IB21001</td>
			<td>Gel transfer</td>
		</tr>
		<tr>
			<td>iBlot2 Transfer Stacks, Nitrocellulose, mini</td>
			<td>Thermo Fisher</td>
			<td>IB23002</td>
			<td>Gel transfer</td>
		</tr>
	</tbody>
</table>

enrichment of detergent-insoluble protein aggregates from human postmortem brain

In dieser Studie beschreiben wir eine abgekürzte Einzelschritt Fraktionierung-Protokoll für die Bereicherung der Waschmittel-unlösliche Aggregate von Post-mortem Gehirn. Das Ionische Waschmittel N-Natriumlaurylsulfat-Sarkosin (Sarkosyl) solubilisiert effektiv nativ gefaltete Proteine im Gehirngewebe, so dass die Anreicherung von Waschmittel-unlösliche Aggregate aus einer breiten Palette von neurodegenerativen Proteinopathies, wie Alzheimer-Krankheit (AD), Parkinson und Amyotrophe Lateralsklerose und Prion-Krankheiten. Menschlichen Kontrolle und Post-mortem Hirngewebe AD wurden homogenisiert und sedimentierte durch Ultrazentrifugation in Anwesenheit von Sarkosyl Waschmittel-unlösliche Aggregate einschließlich pathologischer phosphorylierten Tau, das Herzstück der neurofibrillären bereichern Verwicklungen in AD. Westliche Beflecken, demonstriert die differenzielle Löslichkeit der aggregierten phosphoryliert-Tau und die Waschmittel-lösliche Protein, frühen Endosom Antigen 1 (EEA1) in Steuerung und AD Gehirn. Proteomic Analysen ergaben auch Bereicherung der β-Amyloid (Aβ), Tau, snRNP70 (U1 - 70 K), und Apolipoprotein E (APOE) in die Sarkosyl-unlösliche Bruchteilen von AD Gehirn im Vergleich zu denen der Kontrolle, konsistent mit früheren Gewebe Fraktionierung Strategien . Somit eignet sich dieses einfache Bereicherung-Protokoll für eine Vielzahl von experimentellen Anwendungen reichen von Western blotting und funktionsfähiges Protein Co Aggregation Assays, Massenspektrometrie-basierte Proteomics.

Das abgekürzte einstufigen Sarkosyl-Fraktionierung-Protokoll wurde verwendet, um Waschmittel-unlösliche Aggregate aus Kontrolle und AD Post-mortem Gehirn (Abbildung 1) zu bereichern. Proteine aus TH-S, S1, S2 und P2 Fraktionen wurden gelöst durch SDS-PAGE, für 15 min in Coomassie Blau Fixativ Puffer gefolgt von schonende Färbung mit Coomassie Brilliant Blue G-250 Färbung Puffer fest. Die Wiederfreisetzung Schritt ist optional, da gab es nicht nachweisba...

Watch this Scientific Journal Video about Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain at JoVE.com

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

Eine abgekürzte Fraktionierung-Protokoll für die Bereicherung der Waschmittel-unlösliche Aggregate von Post-mortem Gehirn wird beschrieben.

Anreicherung von Waschmittel-unlösliche Aggregate von Post-mortem Gehirn

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

enrichment-detergent-insoluble-protein-aggregates-from-human

Research

JoVE Journal

Biochemistry

12.0K Aufrufe.  Emory School of Medicine. Eine abgekürzte Fraktionierung-Protokoll für die Bereicherung der Waschmittel-unlösliche Aggregate von Post-mortem Gehirn wird beschrieben.

Serie: Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

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

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

Tandem Affinity Purification von Proteinkomplexen aus eukaryotischen Zellen

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Proteinverdauung, Ultrafiltration und Größe Ausgrenzung Chromatographie, die Isolation der Exosomen aus menschlichem Blutplasma und Serum zu optimieren

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Kalibrierung-kostenlose In-vitro- Quantifizierung von Protein Homo-Oligomerisierung mit kommerziellen Instrumentierung und kostenlose Open Source-Helligkeit-Analyse-Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Hohe Empfindlichkeit Messung der Transkription Faktor-DNA verbindlichen Affinitäten durch wettbewerbsfähige Titration mit Fluoreszenz-Mikroskopie

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Analyse der Proteinfaltung, Transport und Abbau in lebenden Zellen durch radioaktive Puls Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

Nachweis von Protein-Ubiquitinationsstellen durch Peptidanreicherung und Massenspektrometrie

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

Robuste Vergleich der Proteingehalte in Gewebe und in der gesamten Entwicklung mit standardisierten quantitativen Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

Einschaltstige Bereicherung einer TAP-Tagged-Histon-Deacetylase des Filamentous Fungus Aspergillus nidulans für Enzymatische Aktivität Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

Kinase-Inhibitor-Screening in selbstmontierten humanen Protein-Mikroarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...