Diese Arbeit wurde durch Zuschüsse der National Institutes of Health, um VF (NS42599 und GM077569) unterstützt.

1. Vorbereitungen für die Vernetzung <ol><li> Sie werden auf Platte müssen eine ausreichende Anzahl von Zellen zur Isolierung von 500 ug Protein pro Standard-Rohr-Reaktion zu ermöglichen. Eine einzelne Röhre ausreichend sein kann für die Identifizierung von putativen Interaktionspartner eines Proteins von Interesse durch Immunoblot. In diesem Fall Bead gebunden Material kann mit SDS-PAGE-Probenpuffer (Immunpräzipitation) eluiert werden. Für massenspektrometrischen Analyse sollte die Zahl der normalen Reaktionen mindestens zehn Mal erhöht werden und Protein-Komplexe sollten durch out-Wettbewerb mit einem Peptidantigen durch den Antikörper verwendet (Immunaffinitätschromatographie) anerkannt eluiert werden. Diese Strategie ermöglicht die Isolierung von Protein-Komplexen frei von Immunglobulin. </li><li> Die optimale Zelldichte für die Vernetzung zwischen 75% und 90% Konfluenz. In hoher Konfluenz Bedingungen neigen Zellen, die auf jeder anderen Stapel, der nimmt die Zugänglichkeit der zellpermeablen Vernetzer, um alle Zellen. </li><li> Zelltyp und experimentellen Bedingungen sollte auf dem Boden der Zelle Platte markiert werden. Wir routinemäßig auch Kontrollen mit Vehikel. </li><li> Bereiten Sie alle Lösungen außer der DSP-Lösung vor der Vernetzung. <ol><li> Bereiten Sie einen Vorrat an Phosphat-gepufferte Kochsalzlösung mit CaCl 2 0,1 mM und CaCl 2 1 mM (PBS / Ca / Mg) vor Beginn des Experiments. Die Zugabe von Kalzium-und Magnesium-Ionen ist entscheidend für die Adhäsion von Zellen an der Kultur Platte im Verlauf des Experiments. Lagerung bei 4 ° C. </li><li> 50X Komplette Protease Inhibitor Cocktail - 1 Tablette in 1 ml Milli-Q Wasser gelöst. Lagerung bei -20 ° C. </li><li> 20% Triton X-100 wiegt 10g Triton X -100 und verdünnt in einem Gesamtvolumen von 50 ml Milli-Q Wasser. Rock bei 4 ° C über Nacht und bei 4 ° C bis zur Verwendung. Nicht länger als 1 Monat. Verwenden Verdünnungen von diesen 20% Aktien an die Lyse-und IP-Puffer unten beschrieben vorbereiten. </li><li> 50X DSP Quenching-Lösung 1M TRIS auf pH 7,4. Lagerung bei Raumtemperatur. </li><li> Bereiten Sie eine 10-fach Puffer A-Lösung. 10X Puffer A: <ul><li> 50 mL 1 M HEPES </li><li> 150 mL 5 M NaCl </li><li> 10 mL 0,5 M EGTA </li><li> 250 ul 2 M MgCl 2 </li><li> Der pH-Wert auf 7,4 </li><li> Bringen Endvolumen von 500 mL </li></ul> Die 10X Buffer Eine Lösung ist es, 1X Puffer A nach Bedarf verdünnt. 1x Puffer A ist auch in Lysat und Immuno-magnetische-Fällung Puffer verwendet. Buffer A 1X <ul><li> 10 mM Hepes </li><li> 150 mM NaCl </li><li> 1 mM EGTA </li><li> 0,1 mM MgCl 2 </li><li> pH-Wert 7,4 </li></ul></li><li> Lysis Buffer 1X Puffer A + 0,5% Triton X-100. </li><li> Immuno-magnetische Niederschlag Buffer (IP Buffer) 1X Puffer A + 0,1% Triton X-100. </li></ol></li></ol> 2. Vorbereitung der Vernetzungslösung <ol><li> Bereiten Sie die DSP-Lösung unmittelbar vor der Anwendung auf Zellen. DSP ist stark hydrophob und sollte in DMSO vor der Verdünnung in PBS / Ca / Mg-Puffer gelöst werden. Lösen Sie 40 mg des DSP in 1 ml DMSO. Dies macht eine 100 mM Lösung von DSP (Das Molekulargewicht der DSP ist 404,42 g / mol). </li><li> Warm einem geeigneten Volumen PBS / Ca / Mg bis 37 ° C, um eine Verwässerung der DSP / DMSO in PBS zu erleichtern. Volumes für verschiedene Plattengrößen benötigt: 6-Well-Platte 2 mL pro Well 10 cm Platte 10 mL pro Platte 15 cm Platte 20 mL pro Platte </li><li> Add 10 &amp;mgr; l von DSP / DMSO-Stammlösung pro 1 ml warmes PBS / Ca / Mg. Add DSP / DMSO Stammlösung tropfenweise mit wiederholten Mischen, bis alle DSP gelöst hat. Machen Sie eine Control-Lösung von 10 ul DMSO hinzugefügt, um alle 1 ml PBS / Ca / Mg. </li><li> Legen Sie alle PBS / Ca / Mg-Lösungen in einem Eis-Wasser-Bad nicht länger als 10 min. </li></ol> 3. Bereiten Cells für Vernetzung <ol><li> Bereiten Sie ein Eiswasserbad, dass alle Platten der Vernetzung oder der Fahrzeugsteuerung Inkubation passen. </li><li> Nehmen Platten aus dem 37 ° C Inkubator und legen Sie sie sofort in das Eis-Wasser-Bad. </li><li> Wash-Zellen zweimal mit eiskaltem PBS / Ca / Mg. Verwenden Sie das gleiche Volumen, die Sie für den Vernetzer Inkubation verwenden (siehe Abschnitt 2.2). </li><li> Entfernen zweiten Wäsche und fügen entweder Fahrzeugsteuerung oder DSP Vernetzung Pufferlösung. </li><li> Inkubieren auf Eis für zwei Stunden. Sie sollten die Platten etwa alle 20 Minuten, um sicherzustellen, dass alle Zellen, die durch Lösung bedeckt sind. Sie können vielleicht ein wenig DSP aus der Lösung ausfallen. Das ist normal. </li></ol> 4. Die Inaktivierung von DSP Reaction <ol><li> Bereiten Sie eine 1X DSP Abschrecken Lösung von 20 mM Tris pH 7,4 in PBS / Ca / Mg (20 ul 1 M Tris pH 7,4 je 1 ml PBS / Ca / Mg). </li><li> Entfernen Sie das Fahrzeug-Steuerung und Vernetzung Lösungen. </li><li> Add eiskalten Inaktivierung Lösung, Inkubation auf Eis für 15 Minuten. </li></ol> 5. Cell Lysis <ol><li> Während DSP Abschrecken Inkubation, bereiten Sie die Zell-Lyse-Puffer von 0,5% Triton X-100 in Puffer A mit Complete Protease Inhibitor Cocktail. Add 20 uL 50x Stammlösung von Komplett für alle 1 mL Lysepuffer. </li><li> Nach der 15 Minuten DSP Abschrecken Inkubation waschen Sie die Zellen zweimal mit PBS / Ca / Mg. </li><li> Add Lysepuffer + Komplett an Zellen und Fels bei 4 ° C für 30 Minuten. Einige schlugen vor, Volumen Lysepuffer für verschiedene Plattengrößen: <ul><li> 6-well-Platte 0,5 ml pro Vertiefung </li><li> 10 cm Platte 1 mL pro Platte </li><li> 15 cm Platte 1 mL pro Platte </li></ul></li><li> Nach der Lyse mit einem Zellschaber in die Zellen von der Platte zu sammeln. Achten Sie darauf, Lysate auf Eis zu halten, um Protease-Aktivität zu minimieren </li><li> Spin Zelllysate für 15 Minuten bei 4 ° C in einer Tischzentrifuge Mini-Zentrifuge mit hoher Geschwindigkeit (15.000 xg). </li><li> Pipette der Überstand in ein frisches Röhrchen. Verwerfen des Pellets. </li><li> Analysieren Protein-Ebene und fahren Sie mit Immunpräzipitation. </li></ol> 6. Vorbereitung Immuno-magnetische Beads Niederschlag Hinweis: Dieser Schritt ist in der Regel direkt nach Beginn der 2 Stunden Vernetzung Inkubation gestartet. <ol><li> Kombinieren Sie 30 ul Dynal immunomagnetische Kügelchen, 500 ul von IP Buffer (1x Puffer A + 0,1% Triton X-100) und der entsprechenden Menge an Antigen-spezifischen Antikörpern zu Mikrozentrifugenröhrchen Schraubverschluss. Bereiten Antikörper-freie und nicht-spezifischer Antikörper Rohre für negative Kontrollen. Die entsprechende Menge an Antikörper sollte basierend auf einzelne Antigene während separaten Experimenten bestimmt werden. Kennzeichnen Sie alle IP Röhren. </li><li> Lassen Sie die IP-Röhrchen in einem End-over-end Wippe bei Raumtemperatur für 2 Stunden inkubieren. Alternativ inkubieren Sie die IP-Rohre mit dem Antikörper über Nacht bei 4 ° Tag vor der Vernetzung. </li><li> Nach der Inkubation tun, eine schnelle 10 Sekunden Mini-Zentrifugation, um die Perlen am unteren Rand des Röhrchens zu sammeln. </li><li> Schieben Sie die Rohre in die magnetische Halterung, die die magnetischen Kügelchen ziehen wird vom Boden des Röhrchens. Mit den Perlen sicher aus der Art und Weise, können Sie nun ganz einfach wegwaschen alle ungebundenen Antikörper. </li><li> Verwenden Sie eine kleine Bohrung Aspirator Spitze wie eine Spitze für Gel oder p200 Spitze der Inkubationspuffer und alle ungebundenen Antikörper zu entfernen. Sobald die Inkubationsvolumen entfernt in 1 ml IP-Puffer hinzu. </li><li> Cap das Rohr vorsichtig resuspendieren die Perlen, und inkubieren alle IP-Röhrchen bei Raumtemperatur für 5 Minuten mit end-over-end Rotation. </li><li> Wiederholen Sie den 5 Minuten Waschschritt (Schritte 6.3 bis 6.6) noch einmal mit einem frischen Milliliter IP Buffer. </li><li> Die IP-Röhren können in der letzten Waschung Inkubation bleiben, bis das vernetzte Lysat ist bereit, um die Perlen hinzugefügt werden. </li></ol> 7. Inkubieren Vernetzte Lysate mit Immuno-magnetische Beads <ol><li> Nach der letzten Waschung Spin Immuno-magnetische Kügelchen, die in einem Mini-Zentrifuge für 10 Sekunden. Dann fahren Sie mit den Rohren in die magnetische Halterung rutschen. </li><li> Auch mit einem kleinen Bohrung Absaugspitze, entfernen Sie die Inkubationszeit Volumen aus der Tube. Sofort im 500 mL aus vernetztem Zelllysat (vorausgesetzt Lysat bei 1 pg / mL), um die Röhrchen mit immuno-magnetischen Kügelchen. Wenn Sie verwenden möchten Peptid Wettbewerb als negative Kontrolle, sollte eine entsprechende Menge an Peptid an dieser Stelle aufgenommen werden. Frühere Experimente durchgeführt werden, um entsprechende Peptid Wettbewerbsbedingungen zu bestimmen. Alternativ können Sie Lysat freien Immuno-magnetische Kügelchen als negative Kontrolle haben. In diesem Fall sofort hinzufügen 500 ul der Lysate Buffer (1x Puffer A + 0,5% Triton X-100) + Komplette dem Immuno-magnetische Kügelchen. </li><li> Vorsichtig resuspendieren Perlen. Resuspendierten Beads bei 4 ° in einer End-over-end Rotation für 2 Stunden inkubiert werden. </li></ol> 8. Wash Unbound Lysate aus Perlen <ol><li> Sobald Lysat über ausreichende Inkubationszeit hatte, nicht einen schnellen 10 Sekunden Spin in ein Mini-Zentrifuge. Dann schieben Sie die Rohre in die Magnethalterung. Die magnetische Halterung sollte in einem Eisbad für den Rest des Experiments gehalten werden. </li><li> Mit einem kleinen Bohrung Aspirator Spitze, entfernen Sie alle ungebundenen Lysat. Unmittelbar nachdem alle ungebundenen Lysat entfernt 1 mL IP-Puffer. </li><li> Cap der Röhre und sanft resuspendieren Perlen. </li><li> Sobald die Kügelchen sind resuspendiert wiederholen Sie die Perle Pelletierung Schritt (8,1). </li><li> Wieder absaugen alle Inkubationsvolumen und sofort 1 ml IP-Puffer, dann verschließen Sie die Tube. </li><li> Vorsichtig resuspendieren die Perlen in IP Buffer. Inkubieren resuspendierten Beads für 5 Minuten bei 4 ° mit end-over-end zu schaukeln. </li><li> Wiederholen Sie die Perle Waschschritte (8,4 bis 8.6) 4 weitere Male. </li></ol> 9. Denaturieren Probe und Sammeln von Perlen <ol><li> Nach all der Immuno-magnetische Niederschlag Rohren gewaschen wurden, re-Sediment der Perlen (8,1), und schieben Sie Rohre in denMagnethalter. </li><li> Gebundene Proteine, die immuno-magnetische Kügelchen sind in denaturierenden Bedingungen entfernt. Absaugen der letzten IP Buffer waschen und entfernen Sie die IP-Rohr aus dem Magnethalter. </li><li> Add 1x Gel Sample Buffer, um die IP Rohr oberhalb des Wulstes Pellet zu bündeln die Perlen am unteren Rand der Röhre wieder. Die Höhe der Probenpuffer hängt auch Größe des Gels verwendet werden. Möglicherweise müssen Sie leicht auf den Boden der Röhre, um alle der Perlen in der Gel-Probenpuffer resuspendiert bekommen. </li><li> Wiederholen Sie die Denaturierung (9,2 bis 9,3) für jede IP-Rohr. </li><li> Immuno-magnetische Kügelchen in Gel-Probenpuffer resuspendiert werden dann bei 75 ° C für 5 Minuten, um die Denaturierung abzuschließen. </li><li> Sobald die Probe wurde denaturiert es auf einer SDS-PAGE-Gel kann für Western-Blot durchgeführt werden. Entleerte Immuno-Magnetic Beads kann erneut durch Zentrifugation pelletiert und aus der Probe mit dem Magnethalter. </li></ol> 10. Elution von vernetztem Komplexe mit antigenen Peptiden (Immunaffinitätschromatographie). <ol><li> Nach all der Immuno-magnetische Immunpräzipitation Rohre wurden gewaschen, re-Sediment der Perlen (8,1), und schieben Sie Rohre in den Magnethalter. Saugen Sie den Überstand und mit 10 ul Puffer A ergänzt mit dem antigenen Peptid, das durch den Antikörper für Immunisolation von Protein-Komplexen eingesetzt anerkannt. Sie sollten testen Konzentrationen im Bereich von 10 bis 200 pM für die effiziente Elution. </li><li> Inkubieren Sie die Perlen für 2 h bei 4 ° C. </li><li> Legen Sie Perlen in der magnetischen stehen und sorgfältig wieder die 10 ml eluierte Material. Speichern Sie diese Überstand. </li><li> Schnell Waschen der Kügelchen mit Puffer A und entsorgen Sie die Wäsche. Speichern Sie die Perlen. </li><li> Add Gel Sample Buffer, um die gespeicherten Überstand und Perlen zu einer Konzentration von 1X. Inkubieren diese Rohre bei 75 ° C für 5 Minuten. </li><li> Analysieren Perlen-und Peptid-Eluat durch SDS-PAGE. Das Eluat sollte frei von IgG sowohl durch Protein-Färbung von SDS-PAGE oder Immunoblots (Abbildung 1B). Dieses Material frei von IgG ist für Massenspektrometrie. </li></ol><img src="/files/ftp_upload/1855/1855fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Isolierung von AP-3 interagierende Protein-Komplexen und Membranproteine. HEK293-Zellen (A) oder PC12-Zellen (B) wurden entweder in Gegenwart Kontrolle über das Fahrzeug behandelt (DMSO, ungerade Gassen Abb. 1A) oder DSP (auch Bahnen Abb.. 1A oder alle Bahnen in Abb. 1B). Abb.; geklärt Extrakte wurden entweder mit Perlen allein (Abb. 1A, Lanes 3-4), Transferrin-Rezeptor-Antikörper (Abb. 1A, Lanes 5-6) oder AP-3 δ Antikörper (Abb. 1A, Lanes 7-10 inkubiert . 1B, Bahnen 1-10). Immunkomplexe mit SDS-PAGE-Probenpuffer (Abb. 1A) wurden eluiert, Buffer A allein oder (Abb. 1B, Bahnen 1-2), oder Puffer A mit steigenden Konzentrationen des δ antigene Peptid entsprechend den Aminosäuren 680 710 ergänzt der menschlichen δ-Adaptin (NCBI: AAD03777; gi: 1923266) (Abb. 1B, Bahnen 3-10). Dieses Peptid bindet die δ-Antikörper. Nach Peptid Elution wurden die Überstände (S) und Perlen (B) durch Immunoblot (Abb. 1B) analysiert. AP-3, die mit Antikörpern gegen die δ, β3 und σ3 Untereinheiten erkannt wird, Copräzipitate mit den folgenden lösliche Faktoren: Clathrin schwere Kette (CHC), die BLOC-1-Untereinheit pallidin und der HOPS-Komplex Untereinheit vps33b, sowie die Membranproteine ​​Phosphatidylinositol-4-Kinase-Typ-II-alpha (PI4KIIα) und der Zink-Transporter 3 (ZnT3). Beachten Sie das Fehlen von IgG-Maus schweren Ketten in das Peptid eluiert Überstand in Abb.. 1B.

<ol>
	<li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hermansky-Pudlak+syndrome+protein+complexes+associate+with+phosphatidylinositol+4-kinase+type+II+alpha+in+neuronal+and+non-neuronal+cells.">Hermansky-Pudlak syndrome protein complexes associate with phosphatidylinositol 4-kinase type II alpha in neuronal and non-neuronal cells.</a> J Biol Chem. 284, 1790-1802 (2009).</li><li>Lomant, A. J., Fairbanks, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+probes+of+extended+biological+structures:+synthesis+and+properties+of+the+cleavable+protein+cross-linking+reagent+[35S]dithiobis(succinimidyl+propionate.">Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate.</a> J Mol Biol. 104, 243-261 (1976).</li><li>Xiang, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+DSP,+a+reversible+cross-linker,+to+fix+tissue+sections+for+immunostaining,+microdissection+and+expression+profiling.">Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling.</a> Nucleic Acids Res. 32, e185-e185 (2004).</li><li>Craige, B., Salazar, G., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-4-Kinase+Type+II+Alpha+Contains+an+AP-3+Sorting+Motif+and+a+Kinase+Domain+that+are+both+Required+for+Endosome+Traffic.">Phosphatidylinositol-4-Kinase Type II Alpha Contains an AP-3 Sorting Motif and a Kinase Domain that are both Required for Endosome Traffic.</a> Mol Biol Cell. 19, 1415-1426 (2008).</li><li>Newell-Litwa, K., Seong, E., Burmeister, M., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+non-neuronal+functions+of+the+AP-3+sorting+machinery.">Neuronal and non-neuronal functions of the AP-3 sorting machinery.</a> J Cell Sci. 120, 531-541 (2007).</li><li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zinc+Transporter+ZnT3+interacts+with+AP-3+and+it+is+Targeted+to+a+Distinct+Synaptic+Vesicle+Subpopulation.">The Zinc Transporter ZnT3 interacts with AP-3 and it is Targeted to a Distinct Synaptic Vesicle Subpopulation.</a> Mol Biol Cell. 15, 575-587 (2004).</li><li>Trinkle-Mulcahy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+specific+protein+interaction+partners+using+quantitative+mass+spectrometry+and+bead+proteomes.">Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.</a> J Cell Biol. 183, 223-239 (2008).</li></ol>

DSP, eine Membran durchlässig, chemisch reduzierbaren Vernetzer mit einem Spacer-Arm von 12 Å wird verwendet, um transiente Protein-Interaktionen 1,2,3,4 stabilisieren. Hier haben wir diese Strategie mit dem Adapter-Komplex AP-3 ein lösliches Protein-Komplex, und erkennt möglichen Membranproteine ​​in Vesikeln aus Endosomen 5 veranschaulicht. AP-3 bindet selektiv an die Zink-Transporter ZnT3 und der Lipid-Kinase-Phosphatidylinositol-4-Kinase-Typ-II-alpha, aber nicht Transferrin-Rezeptor <su...

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P4417</td>
<td>Dissolve 1 tablet in 200 mL water; add MgCl2 to a final concentration of 1 mM and CaCl2 to a final concentration of 0.1 mM</td>
</tr>
<tr>
<td>Dithiobis (succinimidyl propionate) (DSP)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22585</td>
<td>Moisture sensitive, store in air tight container 4&deg;C</td>
</tr>
<tr>
<td>Dimethyl sulphoxide (DMSO) Hybri-Max</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100, SigmaUltra</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Dynabeads, Sheep anti-Mouse IgG</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.31</td>
<td>Beads are also available as sheep anti-rabbit</td>
</tr>
<tr>
<td>Dyna-Mag-2 magnet</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
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isolation of labile multi-protein complexes by in vivo controlled cellular cross-linking and immuno-magnetic affinity chromatography

Die Dynamik der zellulären Maschinerie häufig auf vorübergehende und / oder schwache Protein-Assoziationen gebaut. Diese geringe Affinität Wechselwirkungen ausschließen strengen Methoden zur Isolierung und Identifizierung von Protein-Netzwerken um ein Protein von Interesse. Der Einsatz von chemischen Vernetzer erlaubt die selektive Stabilisierung der labilen Interaktionen, also unter Umgehung biochemischen Einschränkungen für die Reinigung. Hier präsentieren wir ein Protokoll zugänglich für Zellen in Kultur, dass ein homo Vernetzer verwendet mit einem Spacer-Arm von 12 Å, Dithiobis-(Succinimidyl proprionate) (DSP). DSP wird durch Reduktion einer Disulfidbrücke im Molekül gespalten. Die Vernetzung mit Immunaffinitätschromatographie der Proteine ​​von Interesse mit magnetischen Beads kombiniert ermöglicht die Isolierung von Protein-Komplexen, die sonst nicht überstehen würden Reinigung. Dieses Protokoll ist kompatibel mit Western-Blot-Techniken, und es kann bis zur Proteinidentifizierung skaliert werden durch Massenspektrometrie 1. Stephanie A. Zlatic und Pearl V. Ryder trugen gleichermaßen zu dieser Arbeit.

Watch this Scientific Journal Video about Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography at JoVE.com

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

Die Zelle durchlässig Vernetzer DSP [Dithiobis-(Succinimidylpropionat)] stabilisiert vorübergehend und labile Wechselwirkung<em&gt; In vivo</em&gt;, Die ihre Isolation ermöglicht mit strengen Protein-Komplex Reinigungstechniken. Hier präsentieren wir eine Technik zur Vernetzung von Zellen in Kultur gezüchtet, durch Isolation von Protein-Komplexe durch Immunpräzipitation gefolgt.

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 ...

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

17.2K Aufrufe.  Emory University. Die Zelle durchlässig Vernetzer DSP [Dithiobis-(Succinimidylpropionat)] stabilisiert vorübergehend und labile Wechselwirkung In vivo, Die ihre Isolation ermöglicht mit strengen Protein-Komplex Reinigungstechniken. Hier präsentieren wir eine Technik zur Vernetzung von Zellen in Kultur gezüchtet, durch Isolation von Protein-Komplexe durch Immunpräzipitation gefolgt.

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

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

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization1. Mechanistically, PICUP involves photo-oxidation of Ru2+ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru3+ by irradiation with visible light in the presence of an electron acceptor. Ru3+ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical1,2. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods3,4 include short (&#8804;1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid &beta;-protein variants (A&beta;40 and A&beta;42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

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

Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid &beta;-protein, and calcitonin, and a control peptide growth-hormone releasing factor.

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, ...

Forschung

Biologie

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 ...

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Visualizing Einzelne molekulare Komplexe In Vivo Verwenden der erweiterten Fluoreszenzmikroskopie

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME (Enzyme Linked Immuno Magnetic Elektrochemische) Verfahren zur Mykotoxin-Erkennung

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

In-vivo-Imaging von Intact Drosophila-Larven auf subzellulärer Auflösung

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

Isotherme Titrationskalorimetrie für Mess-Makromolekül-Ligand-Affinität

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Chromatin Isolierung von RNA-Reinigung (Chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

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 ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

AFM-basierte Mapping der elastischen Eigenschaften der Zellwände: auf der Tissue-, Handy-und subzellulärer Beschlüsse

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...