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
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. Co-immunoprecipitatie <ol><li> Zaad HEK293 cellen bij 2 x 10 5 cellen per putje in een 6-well plaat in 2 ml Dulbecco&#39;s gemodificeerd Eagle&#39;s medium (DMEM) supplement met 10% foetaal runderserum (FBS) en 100 U / ml penicilline-streptomycine (Pen-Strep) . </li><li> Kweek cellen gedurende 48 uur bij 37 ° C met 5% CO2. </li><li> Was de cellen met koude fosfaatgebufferde zoutoplossing (PBS) driemaal. Oogst de cellen door centrifugatie bij 500 xg gedurende 5 minuten bij 4 ° C. </li><li> Be...

<ol>
	<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. P., Wellmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold-inducible+RBM3+inhibits+PERK+phosphorylation+through+cooperation+with+NF90+to+protect+cells+from+endoplasmic+reticulum+stress.">Cold-inducible RBM3 inhibits PERK phosphorylation through cooperation with NF90 to protect cells from endoplasmic reticulum stress.</a> FASEB J. 30 (2), 624-634 (2016).</li><li>Fields, S., Song, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+genetic+system+to+detect+protein-protein+interactions.">A novel genetic system to detect protein-protein interactions.</a> Nature. 340 (6230), 245-246 (1989).</li><li>Verhelst, J., De Vlieger, D., Saelens, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-immunoprecipitation+of+the+Mouse+Mx1+Protein+with+the+Influenza+A+Virus+Nucleoprotein.">Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein.</a> J Vis Exp. (98), (2015).</li><li>S&#246;derberg, O., 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=Direct+observation+of+individual+endogenous+protein+complexes+in+situ+by+proximity+ligation.">Direct observation of individual endogenous protein complexes in situ by proximity ligation.</a> Nat Methods. 3 (12), 995-1000 (2007).</li><li>Jarvius, M., 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=In+situ+detection+of+phosphorylated+platelet-derived+growth+factor+receptor+beta+using+a+generalized+proximity+ligation+method.">In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method.</a> Mol Cell Proteomics. 6 (9), 1500-1509 (2007).</li><li>Liu, C. H., 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=Analysis+of+protein-protein+interactions+in+cross-talk+pathways+reveals+CRKL+protein+as+a+novel+prognostic+marker+in+hepatocellular+carcinoma.">Analysis of protein-protein interactions in cross-talk pathways reveals CRKL protein as a novel prognostic marker in hepatocellular carcinoma.</a> Mol Cell Proteomics. 12 (5), 1335-1349 (2013).</li></ol>

Er zijn verschillende voordelen evenals tekortkomingen voor beide methoden. Als relatief nieuwe techniek, een duidelijk voordeel van nabijheid ligatie assay is het haalbaar om eiwit-eiwit interacties op single-cell level in plaats van een partij van heterogene cellen helderen. Beelden met een hoge resolutie en omvang (bijvoorbeeld door confocale microscoop) de mogelijkheid voor de kwantificering door het tellen van enkele fluorescerende vlekken. Daarentegen kan de conventionele combinatie van co-immunoprecipita...

Nucleaire factor 90 (NF90) is een multi-isovorm eiwit met tal van functies, waaronder de reactie op virale infectie, regulering van interleukine-2 post-transcriptie en regulering van miRNA biogenese 1-3. RBM3 een RNA-bindend eiwit, die deze vertaling en miRNA biogenese en kan worden geïnduceerd door verschillende stressoren waaronder hypothermie en hypoxie 4-6. Onlangs vonden we NF90 en RBM3 in een eiwitcomplex 7. De interactie van NF90 en RBM3 essentieel eiwit kinase RNA-achtige endoplasmatisch reticulum kinase (PERK) moduleren in ongevouwen eiwit reactie 7. Zowel NF90 en RBM3 bevinden zich hoofdzakelijk in de kern, maar een klein deel van NF90 en RBM3 shuttle naar het cytoplasma en binden er elkaar voor specifieke functies, bijv PERK activiteit te reguleren. Daarom is het belangrijk om de distributie van NF90-RBM3 interacties visualiseren subcellulair compartiment die kunnen duidenhun verschillende rollen in respectievelijke compartimenten. Decennia geleden, gist twee-hybride (Y2H) ontwikkeld om de interactie te detecteren tussen twee eiwitten 8. Echter, als gevolg van kunstmatige constructie van gefuseerde eiwitten, vals-positieve resultaten van de toepassing van deze methode beperkt. Lange tijd, co-immunoprecipitatie was hoofdtechniek analyseren eiwit-eiwit interacties, vooral bij endogene omstandigheden 9. Aan de gezamenlijk immunogeprecipiteerd eiwitcomplex analyseren Western blot is de meest geschikte techniek, terwijl massaspectrometrie wordt gebruikt bij super gevoeligheid en nauwkeurigheid gewenst. De laatste jaren heeft nabijheid ligatie assay ontwikkeld als een nieuwe methode om eiwit-eiwit interacties in zowel cellen en weefsels te detecteren in situ 10,11. Hier hebben we de meest populaire co-immunoprecipitatie en relatief nieuwe methode nabijheid ligatie assay werkwijze vastleggen NF90-RBM3 interactie in subcellulaire fracties. We hebben ook gesproken over de voordelen en beperkingen van beide technieken.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td>Sigma</td>
			<td>D6429</td>
			<td>High glucose 
			4500 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 1:1000 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 1:1000 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 1:1000 for WB&#160;</td>
		</tr>
		<tr>
			<td>anti-GAPDH antibody</td>
			<td>Abcam</td>
			<td>ab8245</td>
			<td>use 1:1000 for WB&#160;</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-lined secodary antiboy</td>
			<td>Cell Signaling Technology</td>
			<td>#7074</td>
			<td>use 1:5000 for WB&#160;</td>
		</tr>
		<tr>
			<td>anti-mouse HRP secondary antibody</td>
			<td>Carl Roth</td>
			<td>4759.1</td>
			<td>use 1:5000 for WB&#160;</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>1,4-Dithiothreitol (DTT)</td>
			<td>CarlRoth</td>
			<td>6908.1</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>Super RX X-ray film</td>
			<td>Fujifilm</td>
			<td>4741029230</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&#8242;, 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&#160; 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.

Figuur 1 toont aan dat NF90 en RBM3 zowel kerneiwitten en slechts een kleine fractie aanwezig in het cytoplasma. Met name zijn er drie verschillende banden gekleurd positief voor RBM3. De kleinste net onder 20 kDa geeft de juiste maat van RBM3 (het voorspelde molecuulgewicht van RBM3 is 17 kDa). De oorsprong van de twee andere banden nog worden onderzocht. Co-immunoprecipitatie experimenten met RBM3 als lokaas eiwit bleek dat NF90-RBM3 interacties overwegend in de kern e...

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.

Visualisatie van eiwit-eiwit interacties in nucleaire en cytoplasmatische fracties door Co-immunoprecipitatie en<em&gt; In Situ</em&gt; Proximity Ligatie Assay

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

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Biochemistry

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

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