Diese Arbeit wurde durch Zuschüsse AG027818 und AG030709 von NIH / NIA, 2005/2E aus dem Larry L. Hillblom Foundation, IIRG-07-58334 vom Alzheimer Association und 07-65798 vom California Department of Public Health unterstützt.

1. Peptide Vorbereitung <ol><li> Abwiegen ~ 100-200 pg lyophilisierten Peptids unter Verwendung einer Mikrowaage und Transfer in beschriftete, Silizium beschichtet, low-Adsorbens Mikrozentrifugenröhrchen. Hier verwenden wir die menschliche Sequenzen von Aβ40, Aß42, Calcitonin und GRF. </li><li> Hier verwenden wir Peptide mit 1,1,1,3,3,3-Hexafluor-2-propanol (HFIP) vorbehandelt, um eine homogene, Aggregat-freie Präparate zu erhalten. Dieser Schritt ist notwendig, weil vorgeformte Aggregate induzieren schnelle Aggregation von amyloidogenen Proteine, die in schlechten Reproduzierbarkeit unter Versuchen 5 führen. Andere Methoden wie Filtration und SEC können auch verwendet, um Aggregate-freie Präparate für picup 6 erhalten werden. </li><li> Zur Behandlung der Peptide mit HFIP, pre-chill die HFIP Behälter auf Eis in einem Abzug tragen angemessenen Schutz (HFIP ist flüchtig und toxisch). Kühlung von einem 250-ml-Flasche erfordert in der Regel 10-15 min. Add HFIP zu vorgekühlte Röhrchen mit Peptid Lyophilisate zu einem nominalen Peptid-Konzentration von 0,5 mM zu erhalten. </li><li> Beschallen das Peptid-Lösungen in einem Wasser-Ultraschallbad für 5 min bei Raumtemperatur. </li><li> Vortexen und die Röhrchen für 30 min bei Raumtemperatur. </li><li> Gefühlt die Röhrchen auf Eis (für 1 min), und teilen Sie die Lösungen in 10 bis 50-ul-Aliquots in beschrifteten 0,6-ml Low-Adsorbens Rohre. </li><li> Entfernen HFIP durch Verdunstung unter einem leichten Stickstoffstrom, oder lassen Sie die Rohre geöffnet im Abzug über Nacht. Für Übernachtung Verdunstung, statt die offenen Rohre in ein Rack und bedecken sie mit einem großen Blatt Kimwipes, um Staub und Partikel Kontamination zu vermeiden. </li><li> Exsiccate die restlichen HFIP im Vakuum in einem Lyophilisator oder eine Zentrifugenkonzentrator für 30 min, oder in einem Exsikkator befestigt, um ein Vakuum Einlass für 2 h. Das Endprodukt wird ein Peptid Film an der Unterseite des Mikrozentrifugenröhrchen werden. Wenn sie richtig exsiccated, können die Rohre luftdicht über einen längeren Zeitraum (Monate) gelagert werden bei -20 oder -80 ° C. </li></ol> 2. Solubilisierung der HFIP behandelten Peptide und Foto-Vernetzung <ol><li> Vor Solubilisierung der HFIP behandelten Peptide für Vernetzungsreaktionen, muss man die Vernetzung und Abschrecken Reagenzien vorzubereiten. </li><li> Abwiegen Ammoniumpersulfat (APS, Herr 228,2 g / mol) und bereiten eine 20 mM Lösung in 10 mM Natriumphosphat, pH 7,4. Mix mit einem Vortex, bis die Lösung klar ist. </li><li> Bereiten Sie 1 mM Lösung von Tris (2,2-Bipyridyl) dichlororuthenium (II)-Hexahydrat (Rubpy, Herr 748,63 g / mol) in 10 mM Natriumphosphat, pH 7,4. Mix mit einem Vortex und überprüfen vollständige Auflösung. Schützen Sie das Rohr von Licht mit Aluminiumfolie. </li><li> Für SDS-PAGE-Analyse nach Quervernetzung, ist eine bequeme Abschrecken Reagenz 5% β-Mercaptoethanol in 2 × SDS-PAGE-Probenpuffer. Alternativ 1 M Dithiothreitol (DTT, Herr 154,5 g / mol) in deionisiertem Wasser oder einem geeigneten Puffer verwendet werden kann. </li><li> HFIP behandelten Peptid Filme werden in verdünnter NaOH zuerst gelöst und dann Natriumphosphatpuffer wird hinzugefügt, um ~ 30 nM Peptid-Konzentration zu bekommen. Add 60 mM NaOH, gefolgt von VE-Wasser in das Rohr mit dem Peptid-Film, so dass NaOH und Wasser 10 und 45% des letzten Bandes bzw. darstellen. Kratzen Sie die Peptid-Film aus den Innenwänden der Mikrozentrifugenröhrchen mit der Spitze, mischen durch Auf-und Abpipettieren und beschallen für 5 min in einem Wasser-Ultraschallbad. </li><li> Fügen Sie 45% 20 mM Natriumphosphat, pH 7,4, durch Pipettieren gründlich mischen und zentrifugieren bei 16.000 g für 10 min. Beiseite Aliquots von unvernetzten Peptide mit dem Abschrecken Reagenz für negative Kontrollen gemischt. </li><li> Stellen Sie die Kamera-Auslöser-Verzögerung bis 1 s. Bei höheren Belichtungszeiten, können umfangreiche radikalen Reaktionen hervorrufen Proteinabbau. Legen Sie die Kamera-Auslöser. Bestrahlung etwas Zeit brauche, um optimierte, wenn Sie die Methode mit einem neuen Protein sein. </li><li> Ein typisches picup Reaktion wird in einem 20 ul Reaktionsvolumen durchgeführt. Transfer-18 ul der Peptid-Lösung in eine dünnwandige, klar, 0,2-ml PCR-Röhrchen. </li><li> Um die Peptid-Lösung geben, 1 ul Rubpy um 1 ul APS gefolgt und mischen Sie die Reagenzien durch Pipettieren (die Endkonzentration Rubpy und APS in der Reaktionsmischung wird 0,05 und 1 mm, bzw. werden). </li><li> Schnell Platz Reaktionsrohr in einem 1,8-ml-Glasflasche. Legen Sie die Flasche in den Balg vor dem Kameragehäuse befestigt. Befestigen Sie den Objektivschutz und drücken Sie den Auslöser, so dass die Probe für 1 s in den Balg bestrahlt wird. </li><li> Nach Probe-Bestrahlung schnell die Flasche aus dem Balg und PCR-Röhrchen aus dem Fläschchen. Schnell wird die Reaktion durch Zugabe von 1 ul DTT oder 10 ul Reduzierung PAGE Probenpuffer. Wiederholen Sie die Reaktion für jedes Peptid Aliquot. </li><li> Die Reaktionsgemische können nun bei -20 ° C für die Lagerung werden nicht länger als 7 Tage oder auf Eis gehalten vor der Analyse durch SDS-PAGE und Silberfärbung eingefroren. </li></ol> 3. SDS-PAGE und Splitter-Färbungaus vernetztem Peptid-Produkte <ol><li> Routine SDS-PAGE und Silberfärbung werden durchgeführt, um vernetzte Peptide zu visualisieren. </li><li> Legen Sie 5 ul der Reaktionsmischung pro Bahn um ~ 130 pmol Peptid pro Bahn zu erhalten. </li><li> Fügen Sie ähnliche Mengen an unvernetzten Peptide für den Vergleich. Auch eine Standard-Protein-Leiter für visuelle Annäherung mit einem Molekulargewicht von Peptid-Bands. </li><li> Führen Sie das Gel mit einem Standard-Gel-Elektrophorese-Apparatur. Wir nutzen die XCell SureLock Mini-Cell-System von Invitrogen und führen Silber-Färbung nach Invitrogen Veröffentlichung IM-1002, Novex Pre-Cast-Gel-Elektrophorese Guide. </li></ol> Teil 4: Repräsentative Ergebnisse (Abb. 1) <img src="/files/ftp_upload/old/1071_2009_1_19_13/Result no demarcation.png" alt="Abbildung 1" /> Abbildung 1: Silber-gefärbten Polyacrylamidgelen zeigt unvernetzten (-) und Foto-cross-linked (+) GRF, Calcitonin, Aβ40 und Aß42 SDS-PAGE und Silberfärbung analysiert der picup-generated Aβ40-Oligomere zeigen etwa gleiche Bandenintensität von Monomer durch Tetramer, durch einen starken Rückgang in der Fülle der höheren Oligomeren 7 an. Unvernetzte Aβ40 wandert mit einem Herrn im Einklang mit der Monomer 7. Aß42 zeigt eine deutliche Oligomer Größenverteilung. Aß42 Oligomeren umfassen 2-3 Gruppen 8. Die erste Gruppe, Monomer durch Trimer zeigt abnehmender Intensität mit zunehmender Oligomer bestellen. In der zweiten Gruppe, ist ein Gaussian-like Verteilung zwischen Tetramer und Heptamer beobachtet, mit einem Maximum bei Pentamer und Hexamer. Die dritte Gruppe, die hier nicht dargestellt ist, enthält Oligomere von Herrn ~ 30.000-60.000 Da 8. Diese höheren Herrn Oligomere typischerweise beobachtet mit SEC-isolierter LMW Aß42 aber nicht Peptid durch Filtration oder HFIP Behandlung 6 hergestellt. Unvernetzte Aß42 produziert vorwiegend zwei Bands, ein Monomer-Band und einem breiten Trimer / Tetramer Band, die ein Artefakt durch SDS 5,9 induziert wird. Calcitonin ergibt sich ein Oligomer Größenverteilung, die stark abweicht von einem monotonen exponentielle Abnahme in der Region Monomer durch Tetramer, was darauf hindeutet Präexistenz Dimeren, Trimeren und Tetrameren 7. Die Steuerung Polypeptid, GRF, produziert eine monotone Oligomerverteilung von Dimer durch Hexamer, so dass die scheinbare relative Mengen an Oligomeren exponentiell mit zunehmender Molekülmasse. In anderen Experimenten, höhere Oligomere könnten auch visualisiert 7 sein. Die genaue Anzahl und relativen Intensitäten der Oligomer Bands können etwas variieren zwischen Experimente abhängig von der tatsächlichen Protein-Konzentration, die Menge an Protein auf das Gel aufgetragen, und die Zeit für die Entwicklung Schritt in die Silber-Färbung Protokoll verwendet.

<ol>
	<li>Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+study+of+metastable+amyloidogenic+protein+oligomers+by+photo-induced+cross-linking+of+unmodified+proteins.">Structural study of metastable amyloidogenic protein oligomers by photo-induced cross-linking of unmodified proteins.</a> Methods Enzymol. 413, 217-236 (2006).</li><li>Fancy, D. A., Kodadek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+for+the+analysis+of+protein-protein+interactions:+rapid+and+efficient+cross-linking+triggered+by+long+wavelength+light.">Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light.</a> Proc. Natl. Acad. Sci. USA. 96, 6020-6024 (1999).</li><li>Kluger, R., Alagic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+cross-linking+and+protein-protein+interactions&#8212;a+review+with+illustrative+protocols.">Chemical cross-linking and protein-protein interactions&#8212;a review with illustrative protocols.</a> Bioorg. Chem. 32, 451-472 (2004).</li><li>Tomohiro, T., Hashimoto, M., Hatanaka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+chemistry+and+biology:+development+of+multifunctional+photoaffinity+probes.">Cross-linking chemistry and biology: development of multifunctional photoaffinity probes.</a> Chem. Rec. 5, 385-395 (2005).</li><li>Bitan, G., Fradinger, E. A., Spring, S. M., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxic+protein+oligomers&#8212;what+you+see+is+not+always+what+you+get.">Neurotoxic protein oligomers&#8212;what you see is not always what you get.</a> Amyloid. 12, 88-95 (2005).</li><li>Bitan, G., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+aggregate-free,+low+molecular+weight+amyloid-&#946;+for+assembly+and+toxicity+assays.">Preparation of aggregate-free, low molecular weight amyloid-&#946; for assembly and toxicity assays.</a> Methods Mol. Biol. 299, 3-9 (2005).</li><li>Bitan, G., Lomakin, A., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+oligomerization:+prenucleation+interactions+revealed+by+photo-induced+cross-linking+of+unmodified+proteins.">Amyloid &#946;-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins.</a> J. Biol. Chem. 276, 35176-35184 (2001).</li><li>Bitan, G., Kirkitadze, M. D., Lomakin, A., Vollers, S. S., Benedek, G. B., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+(A&#946;)+assembly:+A&#946;40+and+A&#946;42+oligomerize+through+distinct+pathways.">Amyloid &#946;-protein (A&#946;) assembly: A&#946;40 and A&#946;42 oligomerize through distinct pathways.</a> Proc. Natl. Acad. Sci. USA. 100, 330-335 (2003).</li><li>Hepler, R. W., Grimm, K. M., Nahas, D. D., Breese, R., Dodson, E. C., Acton, P., Keller, P. M., Yeager, M., Wang, H., Shughrue, P., Kinney, G., Joyce, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+state+characterization+of+amyloid+&#946;-derived+diffusible+ligands.">Solution state characterization of amyloid &#946;-derived diffusible ligands.</a> Biochemistry (Mosc). 45, 15157-15167 (2006).</li><li>Bitan, G., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+photochemical+cross-linking&#8212;a+new+tool+for+studies+of+metastable,+amyloidogenic+protein+assemblies).">Rapid photochemical cross-linking&#8212;a new tool for studies of metastable, amyloidogenic protein assemblies).</a> Acc. Chem. Res. 37, 357-364 (2004).</li><li>Piening, N., Weber, P., Hogen, T., Beekes, M., Kretzschmar, H., Giese, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-induced+crosslinking+of+prion+protein+oligomers+and+prions.">Photo-induced crosslinking of prion protein oligomers and prions.</a> Amyloid. 13, 67-77 (2006).</li><li>Li, H. T., Lin, X. J., Xie, Y. Y., Hu, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+early+events+of+&#945;-synuclein+oligomerization+revealed+by+photo-induced+cross-linking.">The early events of &#945;-synuclein oligomerization revealed by photo-induced cross-linking.</a> Protein Pept. Lett. 13, 385-390 (2006).</li><li>Vollers, S. S., Teplow, D. B., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Peptide+oligomerization+state+using+rapid+photochemical+crosslinking.">Determination of Peptide oligomerization state using rapid photochemical crosslinking.</a> Methods Mol. Biol. 299, 11-18 (2005).</li></ol>

Picup war ursprünglich eine stabile Protein-Komplexe 2-Studie entwickelt. Die Methode wurde später auf quantitative Studie von metastabilen Amyloid-Protein-Baugruppen, darunter Aß 10, Prionen und krankheits-assoziierte PrP Sc 11 und α-Synuclein 12 aufgebracht. Die wichtigsten Faktoren, die bei der Gestaltung eines picup Experiment werden müssen, sind das Reagenz Stöchiometrie, Bestrahlungszeit und Probenvorbereitung. Die ersten beiden Probleme erfordern empirische Optimierung, während die l...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dolan-Jenner 200-W incandescent lamp</td>
<td>Other</td>
<td>Dolan-Jenner Industries</td>
<td>Model 170-D</td>
<td>The heat generated by the lamp does not affect samples for short incubation periods.</td>
</tr>
<tr>
<td>35-mm SLR camera body</td>
<td>Tool</td>
<td>Pentax</td>
<td>SP500 model</td>
<td>In our settings, a bellows is attached to the body of the camera to provide a convenient chamber for irradiation of the sample placed 10 cm away from the light source.</td>
</tr>
<tr>
<td>Clear, thin walled PCR tubes</td>
<td>Other</td>
<td>Eppendorf</td>
<td>951010006 supplied by Fisher L22-003-24</td>
<td>&nbsp;</td>
<tr>
<td>Glass vials (1.8 mL)</td>
<td>Other</td>
<td>Kimble Chromatography</td>
<td>60940A 2, supplied by Fisher 03-340-60</td>
<td>&nbsp;</td>
<tr>
<td>GRF</td>
<td>Reagent</td>
<td>Bachem</td>
<td>H-3695</td>
<td>&nbsp;</td>
<tr>
<td>HFIP</td>
<td>Reagent</td>
<td>TCI America</td>
<td>H0424</td>
<td>Use in a fume hood.</td>
</tr>
<tr>
<td>A&beta;40 and A&beta;42</td>
<td>Reagent</td>
<td>UCLA Biopolymers Laboratory</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Calcitonin</td>
<td>Reagent</td>
<td>American Peptide</td>
<td>22-1-10</td>
<td>&nbsp;</td>
<tr>
<td>Tris(2,2-bipridyl)dichlororuthenium(II) hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>224758-1G</td>
<td>Vortex until the solution is clear. Cover the RuBpy tube with foil to protect the reagent from ambient light. RuBpy is prepared freshly each time and should be used within 48 h.</td>
</tr>
<tr>
<td>Ammonium persulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-7460</td>
<td>Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h. </td>
</tr>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M7154-25 ML</td>
<td>Can be used when SDS-PAGE analysis is performed.</td>
</tr>
<tr>
<td>Novex Tricine SDS Sample Buffer (2x)</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>LC1676</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell</td>
<td>Tool</td>
<td>Invitrogen</td>
<td>EI0001</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine Gels (10-20%)</td>
<td>Other</td>
<td>Invitrogen</td>
<td>EC6625B0X</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Running Buffer (10x )</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1675</td>
<td>&nbsp;</td>
<tr>
<td>Silver Express Staining Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6100</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

photo-induced cross-linking of unmodified proteins (picup) applied to amyloidogenic peptides

Die Montage der amyloidogene Proteine ​​in toxischen Oligomere ist ein wegweisendes Ereignis in der Pathogenese der Fehlfaltung Erkrankungen, darunter Alzheimer, Parkinson und Huntington-Krankheit, erblich amyotrophe Lateralsklerose, und Typ 2 Diabetes. Aufgrund der metastabilen Charakter dieser Protein-Baugruppen, ist es schwierig, ihre Oligomer Größenverteilung quantitativ zu erfassen mit klassischen Methoden wie Elektrophorese, Chromatographie, Fluoreszenz oder dynamische Lichtstreuung. Oligomere von amyloidogene Proteine ​​existieren als metastabile Mischungen, in denen die Oligomere in Monomere dissoziieren und assoziieren zu größeren Baugruppen gleichzeitig. Picup stabilisiert Oligomer Populationen durch kovalente Vernetzung und wann mit Fraktionierung Methoden, wie Natriumdodecylsulfat-Polyacrylamidgelelektrophorese (SDS-PAGE) oder Größenausschlusschromatographie (SEC) kombiniert wird, liefert picup Schnappschüsse des Oligomers Größe Distributionen, die vor dem Kreuz gab Vernetzung. Daher ermöglicht picup Visualisierung und quantitativen Analyse von metastabilen Protein Populationen und kann zur Montage und entschlüsseln Beziehungen zwischen Sequenz Modifikationen und Oligomerisierung überwachen<sup&gt; 1</sup&gt;. Mechanistisch beinhaltet picup Photo-Oxidation von Ru<sup&gt; 2 +</sup&gt; In einem Tris-(Bipyridyl) Ru (II)-Komplex (Rubpy) zu Ru<sup&gt; 3 +</sup&gt; Durch Bestrahlung mit sichtbarem Licht in Gegenwart von Elektronen-Akzeptor. Ru<sup&gt; 3 +</sup&gt; Ist eine starke Ein-Elektronen-Oxidationsmittel in der Lage zu abstrahieren ein Elektron von einem benachbarten Eiweißmolekül, erzeugt ein Protein, radikale<sup&gt; 1,2</sup&gt;. Radikale sind instabile, hochreaktive Spezies und daher rasch verschwinden durch eine Vielzahl von intra-und intermolekularen Reaktionen. Ein Rest kann nutzen die hohe Energie eines ungepaarten Elektrons mit einem anderen Protein-Monomer Bildung eines dimeren Radikal, das später verliert ein Wasserstoffatom und bildet einen stabilen, kovalent verknüpftes Dimer reagieren. Das Dimer kann dann weiter reagieren durch einen ähnlichen Mechanismus mit Monomeren oder anderen Dimere höherer Ordnung Oligomere bilden. Vorteile der picup im Vergleich zu anderen Foto-oder chemische Vernetzung Methoden<sup&gt; 3,4</sup&gt; Sind kurze (≤ 1 s) Exposition gegenüber nicht-destruktive sichtbares Licht, keine Notwendigkeit für<i&gt; Pre-facto-</i&gt; Änderung der nativen Sequenz und der Länge Null kovalente Vernetzung. Darüber hinaus ermöglicht picup Quervernetzung von Proteinen in weiten pH-und Temperaturbereichen, einschließlich physiologischer Parameter. Hier zeigen wir Anwendung von picup zur Vernetzung von drei amyloidogene Proteine ​​die 40 - und 42-Rückstände Amyloid β-Protein-Varianten (Aβ40 und Aß42) und Calcitonin, und eine Kontroll-Protein, Wachstumshormon-Releasing-Faktor (GRF).

Watch this Scientific Journal Video about Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides at JoVE.com

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Photo-induzierte Quervernetzung von unmodifizierten Proteine ​​(picup) ermöglicht die Charakterisierung des Oligomers Größenverteilung in metastabilen Protein-Mischungen. Wir zeigen, Anwendung picup zu drei Vertreter amyloidogene Peptide der 40 - und 42-Rückstand bildet der Amyloid-β-Protein und Calcitonin, und eine Kontrollgruppe Peptid Wachstumshormon-Releasing-Faktor.

Photo-induzierte Vernetzung von unmodifizierten Proteins (picup) zu amyloidogene Peptide Applied

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

photo-induced-cross-linking-unmodified-proteins-picup-applied-to

Research

JoVE Journal

Biology

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

21.2K Aufrufe.  University of California, Los Angeles. Photo-induzierte Quervernetzung von unmodifizierten Proteine ​​(picup) ermöglicht die Charakterisierung des Oligomers Größenverteilung in metastabilen Protein-Mischungen. Wir zeigen, Anwendung picup zu drei Vertreter amyloidogene Peptide der 40 - und 42-Rückstand bildet der Amyloid-β-Protein und Calcitonin, und eine Kontrollgruppe Peptid Wachstumshormon-Releasing-Faktor.

Serie: Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Elektrophoretische Trennung von Proteinen

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Forschung

Biologie

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

Die Färbung Proteine ​​in Gelen

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

A Lectin HPLC-Methode zur selektiven glykosylierte Peptide aus komplexen biologischen Proben Enrich

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

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 ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Laser Mikrodissektion Angewandt auf Gene Expression Profiling von Untergruppe von Zellen aus dem Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Hochdurchsatz-Screening Quantitative Expression und Aufreinigung rekombinanter zu Disulfid-reichen Proteine ​​Venom Produziert in Applied<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Nicht-chromatographischen Reinigung von rekombinantem Elastin-ähnliche Polypeptide und deren Fusionen mit Peptiden und Proteinen aus<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

Die Verwendung von MALDI-TOF-Massenspektrometrie und einer Kundendatenbank zu den Bakterien Indigene auf eine einzigartige Höhle Umwelt (Kartchner Caverns, AZ, USA) Charakterisieren

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Ein Verfahren zur Auswahl Struktur-Schalt Aptamere zu einem Farbmetrik Gold-Nanopartikeln Assay angewandt

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

Anreicherung von extrazellulären Matrixproteinen aus Gewebe und Verdauung in Peptide für Massenspektrometrie-Analyse

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...