<html> 1. Enzymatische Deglykosylierung <ol><li> Verwenden PCR-Röhrchen, um den Wasserverlust durch Verdunstung zu minimieren. Beschriften Sie ein Set von Röhren 1 bis 7. </li><li> Auftauen 10X G7-Puffer, der 10X-Glykoprotein denaturierenden Puffer, und die 10% NP-40 und klopfen Sie leicht die Rohre, um den Inhalt zu mischen. Halten Sie bei Raumtemperatur. </li><li> Legen Sie die enzymhaltigen Fläschchen auf dem Eis. Versuchen Sie zum Auftauen / Einfrieren-Zyklen zu minimieren. </li><li> Lösen Sie den Inhalt des hCGβ Fläschchen (150 ug) in 600 ul dH 2 O und halten auf dem Eis. </li><li> Bereiten Sie 1 ml 1X G7-Puffer durch Verdünnen des 10fach in dH 2 O. </li><li> Verdünnen 0,5 ul PNGase F in 25 ul 1X G7-Puffer und halten auf dem Eis. </li><li> Bereiten Sie die exoglycosidase Mix (EG-Mix) durch die Kombination von 2 ul jedem α-N-Acetylgalactosaminidase, α1-2 Fucosidase, β1-3 Galactosidase und α1-3, 6 Galactosidase. </li><li> Set up PCR-Röhrchen wie angegeben: </li></ol>ak &quot;&gt; <table border="1"><tbody><tr><td> Tube / Probe # </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> hCGβ (0.25mg/ml) </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> - </td><td> - </td><td> - </td></tr><tr><td> 10X Glycoprotein Denaturierungspuffer </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td></tr><tr><td> dH 2 O </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 9μl </td><td> 9μl </td><td> 9μl </td></tr></tbody></table><ol start="9"><li> Verschließen Sie die Röhrchen, vorsichtig mischen und in dieThermocycler, schließen Sie den Deckel und Denaturierung der Proteine ​​durch Inkubation 10 Minuten bei 94 ° C, mit einem 4 ° C zu halten, gefolgt (mit PCR-Röhrchen in einem Thermocycler stark verhindert die Verdunstung in kleinen Proben). </li><li> Entfernen Sie die Schläuche aus Thermocycler und Zentrifuge zu keiner sichtbaren Kondensation zu entfernen. </li><li> Fügen Sie die folgenden Reagenzien wie angegeben: </li></ol><table border="1"><tbody><tr><td> Sample # </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> 10% NP-40 </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td></tr><tr><td> 10X G7 Buffer </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5 &amp; mu; L </td><td> 2.5μl </td></tr><tr><td> PNGase F 01.50 dil. </td><td> - </td><td> 2μl </td><td> - </td><td> - </td><td> 2μl </td><td> - </td><td> - </td></tr><tr><td> Deglykosylierung Mix </td><td> - </td><td> - </td><td> 2μl </td><td> 2μl </td><td> - </td><td> 2μl </td><td> 2μl </td></tr><tr><td> EG-Mix </td><td> - </td><td> - </td><td> - </td><td> 2μl </td><td> - </td><td> - </td><td> 2μl </td></tr><tr><td> dH 2 O </td><td> 10 &amp;mgr; l </td><td> 8μl </td><td> 8μl </td><td> 6μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td></tr><tr><td> Insgesamt Reaktion vol. </td><td> 25 μ l </td><td> 25 μ l </td><td> 25 μ l </td><td> 25 μ l &lt;/ Td&gt; <td> 25 μ l </td><td> 25 μ l </td><td> 25 μ l </td></tr></tbody></table><ol start="12"><li> Schließen Sie die PCR-Röhrchen mit neuen Kappen (verwerfen die benutzten, da sie nicht richtig passen nach einer Inkubationszeit Zyklus). </li><li> Mischen Sie die Röhrchen durch leichtes Antippen 4 mal und dann drehen Sie den Inhalt nach unten. </li><li> Die Röhrchen in den Thermocycler und bei 37 ° C für 4 Stunden, dann kühlen die Proben auf 4 ° C. </li></ol> 2. SDS-PAGE von Proben deglykosylierter <ol><li> Bereiten Sie 130 ul frisch 3X reduzierenden SDS-Ladepuffer durch Zugabe von 4 pl 1,25 M DTT. </li><li> Add 12,5 ul der vorbereiteten 3X reduzierenden SDS-Ladepuffer zu jeder Probe. </li><li> Schließen Sie die Rohre mit neuen Kappen und klopfen Sie leicht die Rohre zu mischen. </li><li> Die Röhrchen in einem Thermocycler bei 94 ° C für 5 Minuten und dann abkühlen auf 4 ° C. </li><li> Legen Sie 30 ul jeder Probe und 10 ul der Protein-Marker auf einem 10-20% Tris-Glycin-Gel. Save the Rest der Probe für einen Teil 3.1. Legen Sie 10 &amp;mgr; l der ColorPlus Markers. </li><li> Elektrophorese des Gels bei 130 Volt bei Raumtemperatur bis der Farbstoff Front ist nahe der Unterseite des Gels. </li><li> Wenn das Gel beendet ist, entfernen Sie das Gel von der Besetzung und legen Sie sie in eine kleine Plastikbox mit genug Coomasie Bläue, um das Gel zu decken. </li><li> Stain das Gel für 1 Stunde unter leichtem Schütteln. </li><li> Wash Gel dreimal für 30 Minuten in 50 ml entfärben Lösung. </li><li> Notieren Sie sich die Bilder mit einer Weißlicht-Transilluminator oder Scanner. Alternativ kann das Gel zwischen den Blättern der Zellophan in einem Rahmen getrocknet werden. </li></ol> 3. Pro-Q Smaragd 300 für Erkennung von glykosylierten Proteinen in SDS-PAGE-Gelen <ol><li> Parallel mit dem Gel in 2,1), laden den Rest der Proben auf einem 10-20% Tris-Glycin-Gel. Laden 10 &amp; mu; L ColorPlus Markers. </li><li> Während das Gel läuft lösen die Pro-Q Smaragd Reagenz mit DMF und bereiten die Lager Fix, Wash-und Oxidationsmittel-Lösungen für die Pro-Q Smaragd 300 nach dem Produkt-Handbuch mit dem Kit mitgeliefert Fleck. </li><li> Wenn die Elektrophorese beendet ist, entfernen Sie das Gel gegossen und legen Sie sie in einer Kunststoff-Box. </li><li> Fix das Gel durch Zugabe von 100 ml der Fix-Lösung und lassen es über Nacht bei Raumtemperatur unter leichtem Schütteln. </li><li> Wash das Gel mit 100 ml der Waschlösung für 10 bis 20 Minuten bei Raumtemperatur mit gentile Agitation. Wiederholen Sie die Wäsche mit frischer Waschlösung. </li><li> Oxidieren die Kohlenhydrate durch Inkubation des Gels mit leichtem Schütteln für 30 Minuten in 25 ml oxidierende Lösung. </li><li> Wash Gel wie in Schritt 3.5 beschrieben. </li><li> Während das Gel Waschen vorbereiten frischen Pro-Q Smaragd 300 Flecken durch Zugabe von 500 ul des Pro-Q Smaragd 300 Reagenzlösung in Schritt 3,2 bis 25 ml des gelöstenFärbepuffer im Kit enthalten. </li><li> Stain das Gel durch Zugabe von 25 ml des Flecks in Schritt 3,8 vorbereitet und Inkubation im Dunkeln unter leichtem Schütteln für 90 bis 120 Minuten. </li><li> Wiederholen Sie die beiden Waschschritte in Schritt 3.5 beschrieben. </li><li> Notieren Sie sich die Bilder mit einem UV-Transilluminator bei 300 nm. Verwenden Sie das 80 kDa-Marker, die mit Pro-Q Emerald Green gekennzeichnet ist, die UV-Bild auf ein weißes Licht Bild des Gels zeigt die prestained Leiter überlappen. </li><li> Vergleichen Sie die Bilder der Coomasie gefärbten Gel in Schritt 2.10 In der Pro-Q Smaragd gefärbtes Gel aus Schritt 3.11. </li></ol> 4. Repräsentative Ergebnisse Die Änderungen im Protein Migration nach enzymatischer Deglykosylierung sind in Abbildung 2 dargestellt. Vergleichen Sie die Kontrollprobe (Panel A, Spur 1) mit dem PNGase F Behandlung (Entfernung der N-Glykane, Spur 2) und Deglycosylierung Mix (PNGase F; sowie Endo-und Exoglycosidasen zu O-Glykane zu entfernen, Spur 3). Keine weitere Reduktionin der Größe wird nach Verdauung mit zusätzlichen Glycosidasen (Bahn 4) zu sehen. Neben einer Veränderung in der Masse, werden Bänder schärfer als Glykane entfernt werden. Eine Band, die unter dem 17 kDa-Marker (Stern) steht für die vollständig deglykosylierter hCGβ Polypeptid (MW: 16 kDa). Andere Bands können von unvollständigen Deglykosylierung oder aus dem mehrere nicht identifizierte Proteine ​​in der hCGβ Probe (siehe Spur 1) ableiten. Lanes 5 bis 7 (Kontrollen) zeigen die Banden auf, die Glycosidasen. Das Glykoprotein-Färbung von Emerald Green ist in Feld B gezeigt Dieses Reagenz oxidiert und Flecken aller Glykane in ein Eiweißmolekül. Daher ist die Intensität des Signals nimmt mit hCGβ enzymatisch deglykosylierter ist (Spur 1 bis Spur 4). Das Restsignal in Bahnen 3 und 4 zeigen das Vorhandensein von Glykan Motive, die resistent gegen die eingesetzten Enzyme werden. Die zusätzlichen Glycosidasen in Spur 4 verwendet entfernen ein paar zusätzliche Zuckerreste: das Protein Migration ist die gleiche, abereinen leichten Rückgang in der Intensität der Färbung zu erkennen. Beständig Zuckereinheiten wurden nicht in allen Protein-Spezies: einige Bands wurden von Emerald Green (abwesend in die UV-Bild, in Klammern) nachgewiesen, was auf sie ausgiebig waren deglykosylierter. Zusätzliche Daten unterstützen die Schlussfolgerung, dass hCGβ heterogen glykosyliert ist. Die untere Bande auf Bahn 2 (Pfeil) ist schwach auf der Emerald Green Bild, während das obere Band auf Spur 2 ist hell, was darauf hinweist, dass viele Glykane Gruppen noch vorhanden sind. Diese Daten unterstützen die Schlussfolgerung, dass rekombinante hCGβ in Maus-Zellen exprimiert mehrere glycoforms enthält 9. Diese unterschiedlichen glycoforms sind aufgrund der inhärenten Heterogenität der Glykosylierung, wo einige Polypeptide nicht erhalten, ein Glykan in jedem Konsens vor Ort und / oder einige Glykane sind erweitert, während andere sogar auf das gleiche Protein nicht. <img alt="Abbildung 1" src="/files/ftp_upload/3749/3749fig1.jpg" /> Abbildung 1.Typische Glykosylierungsmustern für sezernierte oder Zelloberflächen-Glykoproteine. <img alt="Abbildung 2" src="/files/ftp_upload/3749/3749fig2.jpg" /> Abbildung 2. SDS-PAGE-Gele zeigen enzymatische Deglykosylierung hCGβ. Panel A zeigt ein Coomassie-Blau-Färbung, während Panel B zeigt die Ergebnisse der Pro-Q Smaragd 300 für Visualisierung von glykosylierten Proteinen. Beispiel: 1, hCGβ Kontrolle; 2, PNGase F Verdauung; 3, Deglycosylierung Mix Verdauung; 4, Deglycosylierung Mix plus Exoglycosidasen Verdauung, Proben 5 bis 7 sind die Reagenzien Kontrollen (O-Glyc, O-Glycosidase, GNA, β-N -Acetylglucosaminidase; Gal / Fuc, β1-3 Galactosidase, α1-3, 6 Galactosidase und α1-2 Fucosidase; GalNA, α-N-Acetylglalactosaminidase; Neu, Neuraminidase; PNGase; PNGase F)

<ol>
	<li>Spiro, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+glycosylation:+nature,+distribution,+enzymatic+formation,+and+disease+implications+of+glycopeptide+bonds.">Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds.</a> Glycobiology. 12 (4), 43-56 (2002).</li><li>Ohtsubo, K., Marth, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+in+Cellular+Mechanisms+of+Health+and+Disease.">Glycosylation in Cellular Mechanisms of Health and Disease.</a> Cell. 126, 855-867 (2006).</li><li>Arnold, J. N., Wormald, M. R., Sim, R. B., Rudd, P. M., Dwek, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+glycosylation+on+the+biological+function+and+structure+of+human+immunoglobulins.">The impact of glycosylation on the biological function and structure of human immunoglobulins.</a> Annual Review of Immunology. 25, 21-50 (2007).</li><li>Mitra, N., Sinha, S., Ramya, T. N. C., Surolia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-linked+oligosaccharides+as+outfitters+for+glycoprotein+folding,+form+and+function.">N-linked oligosaccharides as outfitters for glycoprotein folding, form and function.</a> Trends in Biochemical Sciences. 31, 156-163 (2006).</li><li>Carlsen, R. B., Bahl, O. P., Swaminathan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chorionic+gonadotropin.+Linear+amino+acid+sequence+of+the+beta+subunit.">Human chorionic gonadotropin. Linear amino acid sequence of the beta subunit.</a> Journal of Biological Chemistry. 248 (19), 6810-6812 (1973).</li><li>Tarentino, A. L., Plummer, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deglycosylation+of+Asparagine-linked+Glycans+by+PNGaseF.">Deglycosylation of Asparagine-linked Glycans by PNGaseF.</a> Trends in Glycoscience and Glycotechnology. 5 (23), 163-170 (1993).</li><li>Tretter, V., Altmann, F., Marz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine+amidase+F+cannot+release+glycans+with+fucose+attached+&#945;1-3+to+the+asparagine-linked+N-acetylglucosamine+residue.">Peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached &#945;1-3 to the asparagine-linked N-acetylglucosamine residue.</a> European Journal of Biochemistry. 199, 647-652 (1991).</li><li>Koutsioulis, D., Landry, D., Guthrie, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+endo-&#945;-N-acetylgalactosaminidases+with+broader+substrate+specificity.">Novel endo-&#945;-N-acetylgalactosaminidases with broader substrate specificity.</a> Glycobiology. 18 (10), 799-805 (2008).</li><li>Thakur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+the+glycoforms+of+the+intact+alpha+subunit+of+recombinant+human+chorionic+gonadotropin+by+high-resolution+capillary+electrophoresis-mass+spectrometry.">Profiling the glycoforms of the intact alpha subunit of recombinant human chorionic gonadotropin by high-resolution capillary electrophoresis-mass spectrometry.</a> Analytical Chemistry. 81 (2), 8900-8907 (2009).</li><li>Park, S., Lee, M. R., Shin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+microarrays+as+powerful+tools+in+studies+of+carbohydrate-mediated+biological+processes.">Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes.</a> Chemical Communications. 37, 4389-4399 (2008).</li><li>Zachara, N. E., Vosseller, K., Hart, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+analysis+of+proteins+modified+by+O-linked+N-acetylglucosamine.">Detection and analysis of proteins modified by O-linked N-acetylglucosamine.</a> Current Protocols in Molecular Biology. 95, 17-17 (2011).</li><li>Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , (2008).</li></ol>

Die Autoren sind von New England Biolabs, die viele der Reagenzien in diesem Artikel verwendeten produziert beschäftigt.

Die hier beschriebene Methode mit Hilfe enzymatischer Deglykosylierung und SDS-PAGE können wertvolle Informationen über die Glykosylierung eines Proteins von Interesse zu erbringen, während Glykan-spezifische Reagenzien die Interpretation der Daten erleichtern. Dieses Protokoll wird für die ersten Untersuchungen der Protein-Glykosylierung bestimmt, und es ist besonders für sekretorische und Membranglykoproteine ​​aus Säugerzellen geeignet: die Enzyme in diesem Fall gewählt wird speziell entfernen Sie alle N-Glykane und oder N-Glykane plus und die häufigste Zucker Erweiterung und bilden den Kern der O-Glykane. Glycosidasen haben den zusätzlichen Vorteil, dass sie mild, im Vergleich mit chemischen Deglykosylierung Methoden, die Wahrung der Integrität der beiden Zucker-und Protein-Rückgrat. Um die Rate der Belegung (die Aminosäuren glykosyliert sind), das Ausmaß der Glykosylierung oder die Feinstruktur der Glykane, anspruchsvollere Techniken wie m bestimmen Aufklärungass-Spektrometrie, Flüssigchromatographie oder NMR erforderlich sind. Wegen seiner Einfachheit kann in mehreren Schritten in diesem Protokoll angepasst, ersetzt werden, und / oder kombiniert werden, um verschiedene experimentelle Bedürfnisse anzupassen. Um jedoch die Ergebnisse, die eindeutig interpretiert werden können, erhalten, ist es wichtig, seine Stärken und Schwächen zu verstehen. Erstens sind die Spezifität und Reinheit der Glycosidasen entscheidend: nur gut charakterisierten Enzyme getestet, frei zu sein von Proteasen und anderen verunreinigenden Tätigkeit verwendet werden soll. Leider gibt es keine Standard-Enzym-Einheit Definition für Glycosidasen, sollte der Benutzer die entsprechende Substitution nach den Angaben des Herstellers zu bestimmen. Zweitens ist eine sorgfältige Auswahl der Nachweis erforderlich: a) Protein Färbereagenzien sind nur sinnvoll, wenn Deglykosylierung führt zu einer deutlichen Verschiebung in der molekularen Masse. Solch ein eindeutiges Ergebnis wie hier gezeigt, ist nicht immer erhalten. In anderen Fällen haben wir abnormal mi gesehengration nach Deglykosylierung (weit entfernt von dem vorhergesagten Molekulargewicht, oder sogar langsamer Migration). Dieses Phänomen ist nicht gut verstanden, aber es kann gesagt werden, dass jede Änderung in der Migration Beweise dafür, dass das Protein wurde deglykosylierter ist. B) Zucker-Detektion mit Antikörpern einzigartige Herausforderungen Begrenzung ihrer Anwendbarkeit präsentiert werden. Es war sehr schwierig, allgemeine Anti-Glycan Antikörper zu erzeugen, sie sind in der Regel gegen ein komplexes Glycan Ziel, was ihren Einsatz einschränkt angehoben. Darüber hinaus zeigen verschiedene monoklonale anti-Glycan Antikörper unerwünschte Kreuzreaktivität 10. C) Lektine (Proteine ​​mit intrinsischer Zucker Affinität) sind gut für Zucker-Erkennung geeignet, plus sie geben Einblick in Glykanstruktur. Allerdings haben nicht alle von ihnen eine enge Spezifität, und viele sind nur teilweise charakterisiert (das heißt, sie könnten nicht bekannt Affinitäten haben). Als Folge positive Lektin Färbung bietet Indikation, kein Beweis, der die Anwesenheit eines given Zucker. d) Chemische Labeling Kits (basierend auf Perjodat Oxidation von Zuckern) sind die Methode der Wahl für alle Glykoproteine ​​Fleck, und somit gut geeignet, um Deglykosylierung folgen. Bei der Verarbeitung von einer unbekannten Probe, ist es eine gute Übung, Glykoprotein Kontrollen beinhalten. Fetuin ist ein leicht verfügbares N - und O-Glykoprotein, Chorion-Gonadotropin (beide Untereinheiten) ist ebenfalls eine gute Wahl. Rinderserumalbumin (BSA) können als negative Kontrolle verwendet werden. Es sollte jedoch angemerkt, dass einige nicht-glykosylierte Proteine ​​leicht reagieren mit dem Pro-Q Smaragd 300, besonders wenn sie in hohen Konzentrationen eingesetzt werden. Nicht-glykosylierte Molekulargewicht-Standards, wie z. B. die Markers in diesem Protokoll verwendet werden, haben den Vorteil, dass die Anzeige scharfe Banden. Nur durch Zufall ist eines dieser Proteine ​​(80 kDa) reaktiv auf Pro-Q Smaragd 300. Daher könnte der Benutzer wollen ein Glykoprotein Standard Leiter laufen, anstatt wie Candy-Cane aus Invitrogen. Schließlich ist einfach in-Gel-Detektion von nucleocytosolic Glykoproteine ​​(die mit einem einzigen O-GlcNAc modifiziert) mit der Verwendung eines monoklonalen Antikörpers möglich, entlang β-N-Acetylglucosaminidase Spezifität Steuerung 11. Die Beschreibung dieser Technik ist, über die Rahmen dieses Artikels sprengen, aber es sollte erwähnt werden, wie Glycosidasen nützliche Werkzeuge für das Studium der vielen anderen Glykoproteinen und Glycokonjugate in Zellen sind. 12:;: online frei verfügbar auf der NCBI Bookshelf (NBK1908 PMID 20301239 Bookshelf ID;) Eine umfassende Behandlung aller bekannten Formen der Glykosylierung in der zweiten Auflage der &quot;Essentials of Glycobiology&quot; gefunden werden.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Humanes Choriongonadotropin β </td><td> Sigma Aldrich </td><td> C6572 </td><td></td></tr><tr><td> PCR Tubes </td><td> VWR </td><td> 20170-004 </td><td></td></tr><tr><td> PCR Thermocycler </td><td> Applied Biosystems </td><td> 4359659 </td><td></td></tr><tr><td> PNGase F </td><td> New England Biolabs </td><td> P0704 </td><td> Ausgestattet mit Puffer </td></tr><tr><td> Protein Deglycosylierung Mix </td><td> New England Biolabs </td><td> P6039 </td><td> Ausgestattet mit Puffer </td></tr><tr><td> α-N-Acetylglalactosaminidase </td><td> New England Biolabs </td><td> P0734 </td><td> Ausgestattet mit Puffer </td></tr><tr><td> α1-2 Fucosidase </td><td> New England Biolabs </td><td> P0724 </td><td> Ausgestattet mit Puffer </td></tr><tr><td> α1-3, 6 Galactosidase </td><td> New England Biolabs </td><td> P0731 </td><td> Ausgestattet mit Puffer </td></tr><tr><td> β1-3 Galactosidase </td><td> New England Biolabs </td><td> P0726 </td><td> Ausgestattet mit Puffer </td></tr><tr><td> 10X Buffer G7 (500 mM Natriumphosphat) </td><td> New England Biolabs </td><td> --- </td><td> Ausgestattet mit PNGaseF oder Degl Mix </td></tr><tr><td> 10X Glycoprotein Denaturierungspuffer (5% SDS, 400 mM DTT) </td><td> New England Biolabs </td><td> --- </td><td> Ausgestattet mit PNGaseF oder Degl Mix </td></tr><tr><td> 10% NP-40 </td><td> New England Biolabs </td><td> --- </td><td> Ausgestattet mit PNGaseF oder Degl Mix </td></tr><tr><td> 3X SDS-Ladepuffer (187,5 mM Tris-HCl pH 6,8, 6% SDS, 30% Glycerin, 0,03% bromophenol blau) </td><td> New England Biolabs </td><td> B7703 </td><td> Geliefert mit 1,25 M DTT </td></tr><tr><td> ColorPlus Markers </td><td> New England Biolabs </td><td> P7709 </td><td></td></tr><tr><td> 10-20% Tris-Glycin-Multigel </td><td> Cosmo Bio Co. </td><td> DCB-414893 </td><td></td></tr><tr><td> Cassette Elektrophoresekammer </td><td> Cosmo Bio Co. </td><td> DCB-303111 </td><td></td></tr><tr><td> Elektrophorese Power Supply EPS 301 </td><td> GE Healthcare </td><td> 18-1130-01 </td><td></td></tr><tr><td> Pro-Q Smaragd 300 Glykoprotein Stain Kit </td><td> Invitrogen </td><td> P-21857 </td><td></td></tr><tr><td> Brilliant Blue R </td><td> Sigma Aldrich </td><td> B0149 </td><td></td></tr><tr><td> AlphaImager HP System </td><td> Zell Biosciences </td><td> 92-13823-00 </td><td></td></tr></tbody></table>

identification and characterization of protein glycosylation using specific endo- and exoglycosidases

Glykosylierung, die Zugabe von kovalent gebundene Zucker, ist ein wichtiger post-translationale Modifikation von Proteinen, die wesentlich beeinflussen können Prozesse wie Zelladhäsion, molekulare Handels-, Abstands-und Signaltransduktion 1-4. In Eukaryoten sind die häufigsten Glykosylierung Änderungen in den sekretorischen Weg Ergänzungen um einen Konsens Asparaginresten (N-linked), oder an Serin oder Threonin-Reste (O-linked) (Abbildung 1). Initiation von N-Glycan Synthese ist sehr in Eukaryoten konserviert, während die Endprodukte stark variieren kann zwischen verschiedenen Spezies, Geweben oder Proteine. Einige Glykane unverändert bleiben (&quot;high Mannose N-Glykane&quot;) oder werden in den Golgi (&quot;komplexen N-Glykane&quot;) verarbeitet. Größere Vielfalt ist für O-Glykane, die mit einem gemeinsamen N-Acetylgalactosamin (GalNAc)-Rest in tierischen Zellen beginnen, unterscheiden sich aber in niederen Organismen 1 gefunden. ENT &quot;&gt; Die detaillierte Analyse der Glykosylierung von Proteinen ist ein Feld, in sich selbst und erfordert umfangreiche Ressourcen und Expertise, um korrekt ausgeführt. Doch eine Vielzahl der verfügbaren Enzyme, die Zucker zu entfernen (Glycosidasen) möglich macht, eine allgemeine Vorstellung von der Glykosylierung Status haben . ein Protein in einem Standard-Laborbedingungen Hier zeigen die Verwendung von Glycosidasen für die Analyse eines Modells Glykoprotein:. rekombinantes humanes Chorion-Gonadotropin Beta (hCGβ), die zwei N-Glykane und vier O-Glykane 5 trägt die Technik erfordert nur einfache Instrumentierung und typische Verschleißteile, und es kann ohne weiteres auf die Analyse von mehreren Proben Glykoprotein angepasst werden. Mehrere Enzyme können parallel verwendet werden, um ein Glykoprotein, zu studieren. PNGase F ist in der Lage nahezu alle Arten von N-Glycane 6,7 zu entfernen. Für O-Glykane, gibt es keine verfügbaren Enzym, das spalten kann eine intakte Oligosaccharid von the-Protein-Rückgrat. Stattdessen werden O-Glykane von Exoglycosidasen eine kurze Kern, die dann leicht durch O-Glycosidase entfernt getrimmt. Das Protein Deglycosylierung Mix enthält PNGase F, O-Glycosidase, Neuraminidase (Sialidase), β1-4 Galactosidase und β-N-Acetylglucosaminidase. Es wird verwendet, um gleichzeitig zu entfernen N-Glykane und einige O-Glykane 8. Schließlich wurde die Deglycosylierung Mix mit einer Mischung aus anderen Exoglycosidasen (α-N-Acetylgalactosaminidase, α1-2 Fucosidase, α1-3, 6 Galactosidase und β1-3 Galactosidase), die zu entfernen sonst resistenten Monosaccharide, die vorhanden sein in könnte dazu beitragen, ergänzt bestimmte O-Glykane. SDS-PAGE/Coomasie blau wird verwendet, um Unterschiede in der Protein-Migration vor und nach Glycosidase Behandlung visualisieren. Darüber hinaus zeigt eine Zucker-spezifischen Färbemethode, Proq Smaragd-300, verminderte Signal als Glykane succe sindssively entfernt. Dieses Protokoll wird für die Analyse von kleinen Mengen an Glykoprotein (0,5 bis 2 ug) entwickelt, obwohl enzymatische Deglykosylierung hochskaliert werden kann, um größere Mengen an Protein bei Bedarf anzupassen.

Watch this Scientific Journal Video about Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases at JoVE.com

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Mit Hilfe spezifischer Glycosidasen um Zucker aus Glykoproteinen, die durch SDS-PAGE gefolgt zu entfernen, ist eine wertvolle Methode, um glycan Modifikationen am Protein-Proben erkannt und ist eine gute Wahl für die erste Glykobiologie Studien. Veränderungen nach Deglykosylierung kann als Verschiebungen in Gel-Mobilität oder durch Färbung mit glycan empfindliche Reagenzien nachgewiesen werden.

Identifizierung und Charakterisierung von Protein-Glykosylierung mit speziellen Endo-und Exoglycosidasen

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

identification-characterization-protein-glycosylation-using-specific

Research

JoVE Journal

Biology

36.4K Aufrufe.  New England Biolabs. Mit Hilfe spezifischer Glycosidasen um Zucker aus Glykoproteinen, die durch SDS-PAGE gefolgt zu entfernen, ist eine wertvolle Methode, um glycan Modifikationen am Protein-Proben erkannt und ist eine gute Wahl für die erste Glykobiologie Studien. Veränderungen nach Deglykosylierung kann als Verschiebungen in Gel-Mobilität oder durch Färbung mit glycan empfindliche Reagenzien nachgewiesen werden.

Serie: Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identifizierung von Protein-Komplexen mit der quantitativen Proteomik in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Forschung

Biologie

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

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

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

Mit dem GELFREE 8100 Fraktionierung System für Molecular Weight-Based Fraktionierung mit Liquid Phase Wiederherstellung

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

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Identifizierung von Protein Interaktionspartner mittels Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Chemisch-Antikörper blockiert Microarray für Multiplex-High-Throughput-Profiling von spezifischen Protein-Glykosylierung bei Complex Samples

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

Verfahren zur Isolierung und Identifizierung von mRNAs, microRNAs und Protein-Komponenten des Ribonucleoprotein Komplexe aus Zellextrakten mit RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

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 ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Generation, Reinigung und Charakterisierung von Cell-invasive DISC1 Protein Spezies

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Pyrosequencing für Mikrobielle Identifizierung und Charakterisierung

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

Isolierung von spezifischen genomischen Regionen und Identifizierung von assoziierten Moleküle durch enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.