Name: visualization of protein-protein interaction in nuclear and cytoplasmic fractions by co-immunoprecipitation and in situ proximity ligation assay
Uploaded: 2017-01-16T15:00:00.000+00:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay at JoVE.com

This study was supported by the Swiss National Science Foundation (SNSF, 31003A_163305).

1. Coimmunpräzipitation <ol><li> Seed HEK293 Zellen bei 2 × 10 5 Zellen pro Vertiefung in eine 6-Well - Platte in 2 ml nach Dulbecco modifiziertem Eagle-Medium (DMEM) Nahrungsergänzungsmittel mit 10% fetalem Rinderserum (FBS) und 100 U / ml Penicillin-Streptomycin (Pen-Strep) . </li><li> Wachsen Zellen für 48 Stunden bei 37 ° C mit 5% CO 2. </li><li> Wasche die Zellen mit kalter Phosphat-gepufferter Salzlösung (PBS) dreimal. Ernte die Zellen durch Zentrifugation bei 500 xg für 5 min bei 4 ° C. </li><li> Bereiten Sie Kern- und Cytoplasma-Fraktionen kommerziellen Kern und Cytoplasma-Extraktion unter Verwendung von Reagenzien. Folgen Sie Anweisungen des Herstellers mit einigen Modifikationen. <ol><li> Verwenden 3 x 10 6 Zellen für einen Co-Immunpräzipitation Experiment (ungefähr 90% Konfluenz von 3 Vertiefungen einer Platte mit 6 Vertiefungen). In 300 ul kalten Cytoplasma-Extraktionslösung (CER I) und Vortex 15 Sekunden lang mit der höchsten Geschwindigkeit. </li><li> Inkubieren für 30 Minuten auf Eis. Während der Inkubation Vortex für 5 s alle 10 min. </li><li> In 16,5 ul kalt zytoplasmatischen Extraktionslösung II (CER II), Vortex für 5 Sekunden und Inkubation für 5 min auf Eis. </li><li> Rührers 5 sec und Zentrifuge bei 16.000 × g für 5 min bei 4 ° C. </li><li> Den Überstand (Zytoplasmaextrakt) in eine neue vorgekühlte Rohr und auf Eis halten, bis sie verwendet. </li><li> Waschen Sie unlöslichen Pellets (mit Kernen) dreimal mit 1 ml kaltem PBS jedes Mal durch Auf- und Abpipettieren fünfmal. Entfernen PBS nach dem Waschen. </li><li> In 150 ul kalten Kernextraktionslösung (NER), Vortex für 15 Sekunden und Inkubation 1 Stunde auf Eis. Während der Inkubation Vortex für 15 Sekunden und Pipette 10 mal mit einer 200 ul Spitze alle 10 min. </li><li> Vortex für 15 s und Zentrifuge bei 16.000 xg für 10 min bei 4 ° C. </li><li> Den Überstand (Kernextrakt) in eine neue vorgekühlte Röhrchen, auf Eis halten bis zur Verwendung. </li></ol></li><li> Nehmen Sie 10% des Volumens der Kern- und Zytoplasmaextrakte jeweils als Eingänge. </li><li> EINdd kaltem PBS auf die verbleibenden Extrakte auf ein Endvolumen von 1 ml. Halten Sie sich auf Eis bis zum Gebrauch. HINWEIS: Pre-Clearing nicht wesentlich ist, wenn Protein G-konjugierten magnetischen Kügelchen verwendet wird. Wenn jedoch der Hintergrund hoch ist, führen Pre-Clearing von 40 &amp; mgr; l Protein G-konjugierten magnetischen Kügelchen inkubiert (50% Slurry) und 1 ml für 30 min auf einem Rotator Lysat aus diesem Schritt bei 4 ° C verdünnt. Separate Überstand (bereits geklärten Lysat) von Perlen mit Magnethalter. Entsorgen Sie die Perlen. </li><li> Zu kuppeln primäre Antikörper mit Protein G-konjugierten magnetischen Kügelchen, inkubiere 40 &amp; mgr; l Protein G-konjugierten magnetischen Kügelchen (50% ige Aufschlämmung) mit 4 &amp; mgr; g Kaninchen-polyklonalen Anti-RBM3 Antikörper oder Kaninchen-IgG (negative Kontrolle) in 200 &amp; mgr; l PBS plus Tween 20 Puffer (PBST, 0,02% Tween 20) bei Raumtemperatur (RT) für 40 min auf einem Rotator. </li><li> Separate Antikörper-gekoppelten Kügelchen und der Überstand mit einem Magnetträger, und den Überstand verwerfen. Waschen Sie die Perlen einmal mit 200 ul PBST (0,02%). </li><li> 1ml verdünnt Lysaten (oder, falls erforderlich, bereits geklärten Lysate) an die Kügelchen, und über Nacht bei 4 ° C auf einem Rotator inkubiert. </li><li> Am nächsten Tag, trennen Sie die Perlen und Überstand durch einen Magnetträger, und den Überstand verwerfen. Waschen 3 x 10 min mit 0,5 ml PBST (0,02%) für jedes Röhrchen bei 4 ° C auf einem Rotator mit einer festen Drehzahl von 20 Umdrehungen pro Minute. </li><li> Eluieren von Proteinen aus den Perlen durch 40 &amp; mgr; l Probenpuffer Zugabe (1x Handelsprobenpuffer und 50 mM 1,4-Dithiothreitol (DTT)). Hitze bei 70 ° C für 10 min. Spin down und Übertragungsflüssigkeiten in ein neues 1,5-ml-Röhrchen. </li><li> Verdünnte Eingangs Lysate in Probenpuffer (Endkonzentration: 1x Handelsprobenpuffer und 50 mM DTT) und Wärme bei 70 ° C für 10 min. Lasteingänge und immunpräzipitiert Proteine ​​aus dem letzten Schritt zu einer vorgegossenen 4-12% Bis-Tris-Gel. Das Ladevolumen von jeder Vertiefung sollte nicht mehr als 20 &amp; mgr; l nicht überschreiten. Führen Elektrophorese in 1x kommerziellen Laufpuffer auf Eis für 35 min. HINWEIS: Legen Sie nuklearen und Zytoplasmaextrakts in einem Verhältnis von 1: 2 (V / V), die den gleichen Anfangsmenge an Zellen widerspiegelt. </li><li> Transfer-Proteine ​​auf PVDF-Membran in 1x Handelstransferpuffer bei 30 V für 2 h bei 4 ° C. </li><li> Block-Membran mit 5% Magermilch in PBST (0,1% Tween 20) bei RT für 40 min auf einem Schüttler. </li><li> Inkubieren Membran mit primärem Antikörper (beide in einer Verdünnung von 1: 1000) in PBST (0,1%) bei 4 ° C über Nacht. </li><li> Waschen 3 x 10 min mit PBST (0,1%) bei RT auf einem Schüttler. </li><li> Inkubieren Membran mit Meerrettich-Peroxidase (HRP) -konjugiertem Anti-Maus oder Anti-Kaninchen-Sekundärantikörper (beide in einer Verdünnung von 1: 5000) in PBST (0,1%) bei RT für 1 Std. </li><li> Waschen 3 x 10 min mit PBST (0,1%) bei RT auf einem Schüttler. </li><li> Verwenden 0,2 ml verstärkte Chemilumineszenz (ECL) Substratmischung pro cm 2 Membran, inkubiere 5 min bei RT. Vermeiden Sie alles Licht von diesem Schritt ab, außer roten Ampeln. </li><li> Verwerfen Flüssigkeit und belichten zu einem Röntgenfilm in einer Filmkassette. Entwickeln Film einen automatischen Film pr mitHOLZBEARBEITUNGS- Maschine. </li></ol> 2. Immunzytochemie und Proximity Ligation Assay <ol><li> Seed HEK293 - Zellen mit 1,5 × 10 4 Zellen pro Kammer in einem 8-Kammer poly-D-Lysin beschichteten Objektträger in 0,4 ml DMEM mit 10% FBS ergänzt und 100 U / ml Pen-Strep. </li><li> Wachsen Zellen für 48 Stunden bei 37 ° C mit 5% CO 2. </li><li> Absaugen Medium und fixieren Zellen mit 4% Paraformaldehyd (PFA) für 10 min bei RT. </li><li> Absaugen PFA und waschen 3 x 10 min mit 0,5 ml PBS pro Kammer. </li><li> Permeabilisieren und Block-Zellen mit 0,5% Triton X-100 und 5% normalem Ziegenserum (NGS) in PBS bei RT für 1 Std. </li><li> Inkubieren mit primärem Antikörper in 0,1% Triton X-100 und 5% NGS in PBS auf einem Schüttler bei 4 ° C über Nacht. Verdünnte Maus monoklonaler anti-NF90 Antikörper und polyklonalem Kaninchen-anti-RBM3 Antikörper 1: 100 in PBS für Doppelfärbung. Für negative Kontrolle wegzulassen unabhängig eine der beiden primären Antikörper und wegzulassen beide Antikörper in einem dritten Steuer. HINWEIS: Verwenden Sie kommerzielle Blockierung und Verdünnungs Reagenzien verwenden nicht. </li><li> Waschen Sie 3 x 10 min mit 0,5 ml PBS pro Kammer. </li><li> Vorbehaltlich Immunzytochemie oder Nähe Ligatur Assay - Protokolle <ol><li> Immunzytochemie <ol><li> Inkubieren mit 1: 500 verdünnt grün fluoreszierender Farbstoff-gekoppelten anti-Maus und roten Fluoreszenzfarbstoff-gekoppelte Anti-Kaninchen-Sekundärantikörper bei RT für 1 Std. Vermeiden Sie Licht, das von diesem Schritt ab. </li><li> Gegenfärbung Kerne mit 4 &#39;, 6-diamidin-2-phenylindol (DAPI), verdünnt 1: 5.000 in PBS bei RT für 10 min. </li><li> Waschen Sie 3 x 10 min mit 0,5 ml PBS pro Kammer. </li><li> Entfernen Sie die Kammern aus dem Glasträger und trocken. Montieren Sie mit 250 μ, Eindeckmedium pro Folie. </li></ol></li><li> Proximity-Ligation-Assay <ol><li> Bereiten Nähe Ligatur Assay-Sonden. Mischen und Verdünnen zwei Proximity Ligation Assay-Sonden (anti-Maus und Anti-Kaninchen-Sekundär mit verschiedenen Oligonukleotiden gebunden Antikörper, die th ligieren kannrough die Zugabe von zwei weiteren Oligonukleotiden in Ligationslösung) beide 1: 5 in 0,1% Triton X-100 und 5% NGS in PBS, mit einem Gesamtvolumen von 320 &amp; mgr; l für eine 8-Kammerobjektträger (etwa 40 &amp; mgr; l pro cm 2) . 20 min bei RT inkubiert. </li><li> Entfernen Sie die Kammern aus dem Objektträger aus Glas und fügen Sie die verdünnten Sonden. Inkubieren in einer feuchten Inkubator bei 37 ° C für 1 Stunde. </li><li> Bereiten Sie Ligierungslösung. Mischen Sie 8 ul - Ligase, 64 ul 5 x Ligatur Lager und 248 &amp; mgr; l H 2 O. </li><li> Abklopfen die Flüssigkeit aus der Folie, und waschen 2 x 5 min in 1x Waschpuffer A (versehen mit dem Kit). </li><li> Hinzufügen Ligationslösung und Inkubieren in einer feuchten Inkubator bei 37 ° C für 30 min. </li><li> Bereiten Sie Amplifikationslösung. Mix 4 ul - Polymerase, 64 &amp; mgr; l 5x Verstärkung Lager und 252 &amp; mgr; l H 2 O Vermeiden Sie Licht von diesem Schritt ab. </li><li> Abklopfen Flüssigkeit aus dem Schlitten und waschen 2 x 2 min in 1x Wash Buffer A. </li><li> Fügen Sie den Verstärkerlification Lösung und für 100 min bei 37 ° C in einer dunklen Feuchte Inkubator inkubiert. </li><li> Abklopfen die Flüssigkeit aus der Folie, und waschen 2 x 10 min in 1x Waschpuffer B (im Lieferumfang des Kits). Waschen Sie für 1 min in 0,01x Waschpuffer B. </li><li> Trocknen Sie die Rutsche und Halterung mit 250 &amp; mgr; l eines handelsüblichen Montagemedium (mit DAPI) pro Folie. </li></ol></li></ol></li><li> Untersuchen Sie Fluoreszenzsignale unter einem Mikroskop mit einem 10x Okular Objektiv und 20X Objektiv verwendet wird. Erwerben Sie Bilder von einer CCD-Kamera. </li></ol>

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	<li>Pati&#241;o, C., Haenni, A. L., Urcuqui-Inchima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF90+isoforms,+a+new+family+of+cellular+proteins+involved+in+viral+replication?.">NF90 isoforms, a new family of cellular proteins involved in viral replication?.</a> Biochimie. 108, 20-24 (2015).</li><li>Shim, J., Lim, H., R Yates, J., Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+export+of+NF90+is+required+for+interleukin-2+mRNA+stabilization.">Nuclear export of NF90 is required for interleukin-2 mRNA stabilization.</a> Mol Cell. 10 (6), 1331-1344 (2002).</li><li>Sakamoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NF90-NF45+complex+functions+as+a+negative+regulator+in+the+microRNA+processing+pathway.">The NF90-NF45 complex functions as a negative regulator in the microRNA processing pathway.</a> Mol Cell Biol. 29 (13), 3754-3769 (2009).</li><li>Dresios, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+stress-induced+protein+Rbm3+binds+60S+ribosomal+subunits,+alters+microRNA+levels,+and+enhances+global+protein+synthesis.">Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis.</a> Proc Natl Acad Sci. 102 (6), 1865-1870 (2005).</li><li>Danno, S., Itoh, K., Matsuda, T., Fujita, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+expression+of+mouse+Rbm3,+a+cold-shock+protein,+in+Sertoli+cells+of+cryptorchid+testis.">Decreased expression of mouse Rbm3, a cold-shock protein, in Sertoli cells of cryptorchid testis.</a> Am J Pathol. 156 (5), 1685-1692 (2000).</li><li>Wellmann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen-regulated+expression+of+the+RNA-binding+proteins+RBM3+and+CIRP+by+a+HIF-1-independent+mechanism.">Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1-independent mechanism.</a> J Cell Sci. 117 (Pt 9), 1785-1794 (2004).</li><li>Zhu, X., Zelmer, A., Kapfhammer, J. 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The authors have nothing to disclose.

Es gibt mehrere Vorteile sowie Nachteile für beide Methoden. Als eine relativ neue Technik, ein offensichtlicher Vorteil Proximity Ligation Assay ist die Durchführbarkeit Protein-Protein-Wechselwirkungen auf Einzelzellebene statt einer Charge von heterogenen Zellen aufzuklären. Bilder mit hoher Auflösung und Größe (zB durch konfokale Mikroskop) bieten die Möglichkeit zur Quantifizierung von einzelnen fluoreszierenden Flecken zu zählen. Im Gegensatz dazu kann die herkömmliche Kombination von Co-Immunpr?...

Nuclear factor 90 (NF90) ist ein Multi-Isoform - Proteins mit zahlreichen Funktionen , einschließlich der Reaktion auf eine virale Infektion, die Regulierung von Interleukin-2 post-Transkription und Regulierung der miRNA Biogenese 1-3. RBM3 ist ein RNA-bindendes Protein, involviert in Translation und miRNA Biogenese und kann durch verschiedene Stressoren einschließlich Hypothermie und Hypoxie 4-6 induziert werden. Vor kurzem fanden wir NF90 und RBM3 in einem Proteinkomplex 7. Das Zusammenspiel von NF90 und RBM3 ist essentielles Protein - Kinase - RNA-ähnliche Endoplasmatischen Retikulum - Kinase (PERK) Aktivität in ungefalteten Protein - Reaktion 7 zu modulieren. Sowohl NF90 und RBM3 befinden sich überwiegend im Kern aber einem geringen Anteil an NF90 und RBM3 Shuttle in das Zytoplasma und binden dort miteinander für bestimmte Funktionen, beispielsweise PERK Aktivität zu regulieren. Daher ist es wichtig, die Verteilung von NF90-RBM3 Wechselwirkungen in dem subzellulären Kompartiment zu visualisieren, die anzeigen kannihre verschiedenen Rollen in den jeweiligen Fach. Vor Jahrzehnten, Hefe - Zwei - Hybrid (Y2H) wurde die Wechselwirkung zwischen zwei Proteinen 8 zu erkennen , entwickelt. Aufgrund künstliche Konstruktion von Fusionsproteinen, falsch-positive Ergebnisse haben die Anwendung dieses Verfahrens beschränkt. Für eine lange Zeit, Co-Immunpräzipitation war das Haupt Technik Protein-Protein - Wechselwirkungen, insbesondere in endogener Bedingungen 9 zu analysieren. Um die Co-immunpräzipitiert Proteinkomplex zu analysieren, Western-Blot ist die bequemste Methode, während der Massenspektrometrie verwendet wird, wenn Super Empfindlichkeit und Genauigkeit erwünscht sind. In den letzten Jahren Proximity Ligation Assay wurde als ein neues Verfahren entwickelt worden , Protein-Protein - Wechselwirkungen in beiden Zellen und Gewebe in situ 10,11 zu erkennen. Hier verglichen wir die beliebtesten Coimmunpräzipitation Verfahren und relativ neuartige Nähe Ligatur Testverfahren in NF9 Erfassung0-RBM3 Interaktion in subzellulären Fraktionen. Wir diskutierten auch die Vorteile und Grenzen beider Techniken.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>Sigma</td><td>D6429</td><td>High glucose&lt;br/&gt; 4,500 mg/L</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco, Thermo Fisher Scientific</td><td>10270106</td><td></td></tr><tr><td>Penicillin-Streptomycin (PenStrep)</td><td>BioConcept</td><td>4-01F00-H</td><td></td></tr><tr><td>NE-PER Nuclear and Cytoplasmic Extraction Reagents</td><td>Thermo Fisher Scientific</td><td>78833</td><td></td></tr><tr><td>1,4-Dithiothreitol (DTT)</td><td>Carl Roth</td><td>6908.3</td><td></td></tr><tr><td>Dynabeads Protein G</td><td>Novex, Thermo Fisher Scientific</td><td>10003D</td><td></td></tr><tr><td>DRBP76 (NF90/NF110) antibody</td><td>BD Transduction Laboratories</td><td>612154</td><td>use 1:1,000 for WB and 1:100 for ICC/PLA</td></tr><tr><td>RBM3 antibody</td><td>ProteinTech</td><td>14363-1-AP</td><td>use 1:1,000 for WB and 1:100 for ICC/PLA</td></tr><tr><td>Lamin A/C antibody</td><td>Cell Signaling Technology</td><td>#2032</td><td>use 1:1,000 for WB&amp;nbsp;</td></tr><tr><td>anti-GAPDH antibody</td><td>Abcam</td><td>ab8245</td><td>use 1:1,000 for WB&amp;nbsp;</td></tr><tr><td>normal rabbit IgG</td><td>Santa Cruz</td><td>sc-2027</td><td></td></tr><tr><td>anti-rabbit IgG, HRP-linked secondary antibody</td><td>Cell Signaling Technology</td><td>#7074</td><td>use 1:5,000 for WB&amp;nbsp;</td></tr><tr><td>anti-mouse HRP secondary antibody</td><td>Carl Roth</td><td>4759.1</td><td>use 1:5,000 for WB&amp;nbsp;</td></tr><tr><td>Clarity Western ECL Blotting Substrate</td><td>Bio-Rad</td><td>#1705060</td><td></td></tr><tr><td>NuPAGE Novex 4-12% Bis-Tris Gel</td><td>Novex, Thermo Fisher Scientific</td><td>NP0321BOX</td><td></td></tr><tr><td>NuPAGE LDS Sample Buffer (4x)</td><td>Novex, Thermo Fisher Scientific</td><td>NP0007</td><td></td></tr><tr><td>NuPAGE MES SDS Running Buffer (20x)</td><td>Novex, Thermo Fisher Scientific</td><td>NP0002</td><td></td></tr><tr><td>NuPAGE Transfer Buffer (20x)</td><td>Novex, Thermo Fisher Scientific</td><td>NP00061</td><td></td></tr><tr><td>Amersham Hypond P 0.2 PVDF membrane</td><td>GE Healthcare Life Sciences</td><td>10600021</td><td></td></tr><tr><td>Poly-D-Lysine 8 Well Culture Slide</td><td>Corning BioCoat</td><td>354632</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Normal goat serum (NGS)</td><td>Gibco, Thermo Fisher Scientific</td><td>PCN5000</td><td></td></tr><tr><td>Goat anti-mouse IgG (H+L Antibody), Alexa Fluor 488 conjugate</td><td>Thermo Fisher Scientific</td><td>A-11001</td><td></td></tr><tr><td>Goat anti-rabbit IgG (H+L Antibody), Alexa Fluor 568 conjugate</td><td>Thermo Fisher Scientific</td><td>A-11011</td><td></td></tr><tr><td>4&amp;prime;, 6-Diamidin-2-phenylindol (DAPI)</td><td>Sigma</td><td>D9542</td><td></td></tr><tr><td>Duolink PLA probe Anti-mouse PLUS</td><td>Sigma</td><td>DUO92001</td><td></td></tr><tr><td>Duolink PLA&amp;nbsp; probe Anti-rabbit MINUS</td><td>Sigma</td><td>DUO92005</td><td></td></tr><tr><td>Duolink Detection Reagents Red</td><td>Sigma</td><td>DUO92008</td><td></td></tr><tr><td>Duolink Wash Buffers Fluorescence</td><td>Sigma</td><td>DUO82049</td><td></td></tr><tr><td>Duolink Mounting Medium with DAPI</td><td>Sigma</td><td>DUO82040</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Sigma</td><td>81381</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>AX-70</td><td></td></tr><tr><td>CCD camera</td><td>SPOT</td><td>Insight 2MP Firewire</td><td></td></tr><tr><td>X-ray film</td><td>Fujifilm</td><td>Super RX</td><td></td></tr><tr><td>Film processing machine</td><td>Fujifilm</td><td>FPM-100A</td><td></td></tr></tbody></table>

visualization of protein-protein interaction in nuclear and cytoplasmic fractions by co-immunoprecipitation and in situ proximity ligation assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Abbildung 1 zeigt , dass NF90 und RBM3 sind beide Kernproteine und nur ein kleiner Teil ist in das Zytoplasma. Bemerkenswert ist, gibt es drei verschiedene Bands für RBM3 positiv gefärbt. Die kleinste knapp unter 20 kDa die richtige Größe der RBM3 reflektiert (das vorhergesagte Molekulargewicht von RBM3 ist 17 kDa). Der Ursprung der beiden anderen Bänder noch untersucht werden. Co-Immunpräzipitationsexperimenten mit RBM3 als Köder-Protein ergab, dass NF90-RBM3 Wec...

Watch this Scientific Journal Video about Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay at JoVE.com

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Visualisierung von Protein-Protein-Wechselwirkung in Kern- und Cytoplasma-Fraktionen durch Co-Immunpräzipitation und<em&gt; In Situ</em&gt; Nähe Ligatur Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

visualization-protein-protein-interaction-nuclear-cytoplasmic

Research

JoVE Journal

Biochemistry

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

12.7K Aufrufe.  University of Basel. Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Serie: Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

Nachweis und Visualisierung von DNA-schadeninduzierten Proteinkomplexen in Suspensionszellkulturen unter Verwendung des Proximity Ligation Assays

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Forschung

Biochemie

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Erkennung von Heterodimerization Protein-Isoformen, die mit einem in Situ Nähe Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Quantifizierung der Protein-Interaktionsnetzwerkdynamik mit Multiplex-Co-Immunopräzipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

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

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

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

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

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

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

In Situ Detektion von Ribonucleoprotein Complex Assembly in der C. elegans Germline mit Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

Entwicklungsbiologie

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Detektion<em&gt; In-situ-</em&gt; Protein-Protein-Komplexe bei der<em&gt; Drosophila</em&gt; Larven Neuromuskuläre Junction Mit Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Neurowissenschaften

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Imaging Protein-Protein-Wechselwirkungen In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Biologie

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

In Situ-Überwachung von transient gebildeten molekularen Chaperon-Baugruppen in Bakterien, Hefe und menschlichen Zellen

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium and lipids) between the juxtaposed organelles without involving membrane fusion. MCSs are essential for cellular homeostasis, and their functions are ensured by the resident components, which often exist as multimeric protein complexes. MCSs often involve the endoplasmic reticulum (ER), a major site of lipid synthesis and cellular calcium storage, and are particularly important for organelles, such as the mitochondria, which are excluded from the classical vesicular transport pathways. In the last years, MCSs between the ER and mitochondria have been extensively studied, as their functions strongly impact cellular metabolism/bioenergetics. Several proteins have started to be identified at these contact sites, including membrane tethers, calcium channels, and lipid transfer proteins, thus raising the need for new methodologies and technical approaches to study these MCS components. Here, we describe a protocol consisting of combined technical approaches, that include proximity ligation assay (PLA), mitochondria staining, and 3D imaging segmentation, that allows the detection of proteins that are physically close (&gt;40 nm) to each other and that reside on the same membrane at ER-mitochondria MCSs. For instance, we used two ER-anchored lipid transfer proteins, ORP5 and ORP8, which have previously been shown to interact and localize at ER-mitochondria and ER-plasma membrane MCSs. By associating the ORP5-ORP8 PLA with cell imaging software analysis, it was possible to estimate the distance of the ORP5-ORP8 complex from the mitochondrial surface and determine that about 50% of ORP5-ORP8 PLA interaction occurs at ER subdomains in close proximity to mitochondria.

The need for new approaches to study membrane contact sites (MCSs) has grown due to increasing interest in studying these cellular structures and their components. Here, we present a protocol that integrates previously available microscopy technologies to identify and quantify intra-organelle and inter-organelle protein complexes that reside at MCSs.

Visualisierung und Quantifizierung endogener intraorganeller Proteininteraktionen an ER-Mitochondrien-Kontaktstellen durch Proximity-Ligation-Assays

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium ...

<meta charset="utf8"></head><body>Bewertung der Proteininteraktion und -lokalisation bei DNA-Schäden

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that cooperate in this process allows the identification of alternative targets for therapeutic intervention in several diseases, such as cancer, aging-related diseases, and chronic inflammation. The Proximity Ligand Assay (PLA) emerged as a tool for estimating interaction between proteins as well as spatial proximity among organelles or cellular structures and allows the temporal localization and co-localization analysis under stress conditions, for instance. The method is simple because it is similar to conventional immunofluorescence and allows the staining of an organelle, cellular structure, or a specific marker such as mitochondria, endoplasmic reticulum, PML bodies, or DNA double-strand marker, yH2AX simultaneously. The phosphorylation of the S139 at Histone 2A variant, H2AX, then referred to as yH2AX, is widely used as a very sensitive and specific marker of DNA double-strand breaks. Each focus of yH2AX staining corresponds to one break in DNA that occurs a few minutes after the damage. The analysis of changes in yH2AX foci is the most common assay for studying if the protein of interest is implicated in DNA damage response (DDR). Whether a direct role in the DNA damage site is expected, fluorescence microscopy is used to verify the colocalization of the protein of interest with yH2AX foci. However, except for the new super-resolution fluorescence methods, to conclude, the local interaction with DNA damage sites can be a little subjective. Here, we show an assay to evaluate the localization of proteins in the DDR pathway using yH2AX as a marker of the damage site. This assay can be used to characterize the temporal localization under different insults that cause DNA damage.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Kombination von Proximity-Liganden-Assay mit Gamma-H2AX-Färbung zur Charakterisierung von Proteininteraktionen in der DNA-Schadensantwort

Here, we present a simple and rapid protocol for the detection of protein interaction at DNA damage sites.

Proximity-Liganden-Assay zur Lokalisierung von Proteinen in DNA-Schadensstellen

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that ...

Visualisierung von Protein-Protein-Wechselwirkung in Kern- und Cytoplasma-Fraktionen durch Co-Immunpräzipitation und

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