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

1. Co-immunoprecipitation
<ol>
	<li>Seed HEK293 cells at 2 x 105 cells per well in one 6-well plate in 2 ml Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplement with 10% fetal bovine serum (FBS) and 100 U/mL Penicillin-Streptomycin (Pen-Strep).</li>
	<li>Grow cells for 48 hr at 37 &#176;C with 5% CO2.</li>
	<li>Wash cells with cold phosphate-buffered saline (PBS) three times. Harvest cells by centrifugation at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Prepare nuclear and cytoplasmic fractions using commercial nuclear and cytoplasmic extraction reagents. Follow manufacturer&#39;s instruction with some modifications.
	<ol>
		<li>Use 3 x 106 cells for one co-immunoprecipitation experiment (approximately with 90% confluency from 3 wells of one 6-well plate). Add 300 &#181;l cold cytoplasmic extraction solution (CER I) and vortex for 15 sec at the highest speed.</li>
		<li>Incubate for 30 min on ice. During incubation, vortex for 5 s every 10 min.</li>
		<li>Add 16.5 &#181;l cold cytoplasmic extraction solution II (CER II), vortex for 5 sec and incubate for 5 min on ice.</li>
		<li>Vortex for 5 sec and centrifuge at 16,000 x g for 5 min at 4 &#176;C.</li>
		<li>Transfer supernatant (cytoplasmic extract) into a new pre-chilled tube, and keep on ice until used.</li>
		<li>Wash insoluble pellets (containing nuclei) three times with 1 ml cold PBS each time by pipetting up and down five times. Remove PBS after wash.</li>
		<li>Add 150 &#181;l cold nuclear extraction solution (NER), vortex for 15 sec and incubate 1 hr on ice. During incubation, vortex for 15 sec and pipette 10 times with a 200 &#181;l tip every 10 min.</li>
		<li>Vortex for 15 s and centrifuge at 16,000 x g for 10 min at 4 &#176;C.</li>
		<li>Transfer supernatant (nuclear extract) into a new pre-chilled tube, keep on ice until use.</li>
	</ol>
	</li>
	<li>Take out 10% of the volume of nuclear and cytoplasmic extracts each as inputs.</li>
	<li>Add cold PBS to the remaining extracts to a final volume of 1 ml. Keep on ice until use. 
	NOTE: Pre-clearing is not essential when using Protein G-conjugated magnetic beads. However, if the background is high, perform pre-clearing by incubating 40 &#181;l Protein G-conjugated magnetic beads (50% slurry) and 1 ml diluted lysate from this step at 4 &#176;C for 30 min on a rotator. Separate supernatant (pre-cleared lysate) from beads with magnetic rack. Discard the beads.</li>
	<li>To couple primary antibody with Protein G-conjugated magnetic beads, incubate 40 &#181;l Protein G-conjugated magnetic beads (50% slurry) with 4 &#181;g rabbit polyclonal anti-RBM3 antibody or rabbit IgG (negative control) in 200 &#181;l PBS plus Tween 20 buffer (PBST, 0.02% Tween 20) at room temperature (RT) for 40 min on a rotator.</li>
	<li>Separate antibody-coupled beads and supernatant with a magnetic rack, and discard the supernatant. Wash the beads once with 200 &#181;l PBST (0.02%).</li>
	<li>Add 1 ml diluted lysates (or if necessary, pre-cleared lysates) to the beads, and incubate on a rotator at 4 &#176;C overnight.</li>
	<li>On the next day, separate the beads and supernatant by a magnetic rack, and discard the supernatant. Wash 3 x 10 min with 0.5 ml PBST (0.02%) for each tube at 4 &#176;C on a rotator with a fixed speed of 20 rpm.</li>
	<li>Elute proteins from the beads by adding 40 &#181;l sample buffer (1x commercial sample buffer and 50 mM 1,4-dithiothreitol (DTT)). Heat at 70 &#176;C for 10 min. Spin down and transfer liquids to a new 1.5 ml tube.</li>
	<li>Dilute input lysates in sample buffer (final concentration: 1x commercial sample buffer and 50 mM DTT) and heat at 70 &#176;C for 10 min. Load inputs and immunoprecipitated proteins from the last step to a precast 4-12% bis-Tris gel. The loading volume of each well should not exceed 20 &#181;l. Perform electrophoresis in 1x commercial running buffer on ice for 35 min. 
	NOTE: Load nuclear and cytoplasmic extracts in a ratio of 1:2 (V/V), which reflects the same initial amount of cells.</li>
	<li>Transfer proteins to PVDF membrane in 1x commercial transfer buffer at 30 V for 2 hr at 4 &#176;C.</li>
	<li>Block membrane with 5% skimmed milk in PBST (0.1% Tween 20) at RT for 40 min on a shaker.</li>
	<li>Incubate membrane with primary antibodies (both diluted in 1:1,000) in PBST (0.1%) at 4 &#176;C overnight.</li>
	<li>Wash 3 x 10 min with PBST (0.1%) at RT on a shaker.</li>
	<li>Incubate membrane with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (both diluted in 1:5,000) in PBST (0.1%) at RT for 1 hr.</li>
	<li>Wash 3 x 10 min with PBST (0.1%) at RT on a shaker.</li>
	<li>Use 0.2 ml enhanced chemiluminescence (ECL) substrate mixture per cm2 membrane, incubate at RT for 5 min. Avoid all light from this step onwards except red lights.</li>
	<li>Discard liquid and expose to an X-ray film in a film cassette. Develop film using an automatic film processing machine.</li>
</ol>
2. Immunocytochemistry and Proximity Ligation Assay
<ol>
	<li>Seed HEK293 cells at 1.5 x 104 cells per chamber in one 8-chamber poly-D-lysine coated slide in 0.4 ml DMEM supplemented with 10% FBS and 100 U/ml Pen-Strep.</li>
	<li>Grow cells for 48 hr at 37 &#176;C with 5% CO2.</li>
	<li>Aspirate medium, and fix cells with 4% paraformaldehyde (PFA) for 10 min at RT.</li>
	<li>Aspirate PFA, and wash 3 x 10 min with 0.5 ml PBS per chamber.</li>
	<li>Permeabilize and block cells with 0.5% Triton X-100 and 5% normal goat serum (NGS) in PBS at RT for 1 hr.</li>
	<li>Incubate with primary antibodies in 0.1% Triton X-100 and 5% NGS in PBS on a shaker at 4 &#176;C overnight. Dilute mouse monoclonal anti-NF90 antibody and rabbit polyclonal anti-RBM3 antibody 1:100 in PBS for double-staining. For negative control, omit independently either of the two primary antibodies, and omit both antibodies in a third control. 
	NOTE: Do not use commercial blocking and diluting reagents.</li>
	<li>Wash 3 x 10 min with 0.5 ml PBS per chamber.</li>
	<li>Subject to immunocytochemistry or proximity ligation assay protocols
	<ol>
		<li>Immunocytochemistry
		<ol>
			<li>Incubate with 1:500 diluted green fluorescent dye-coupled anti-mouse and red fluorescent dye-coupled anti-rabbit secondary antibodies at RT for 1 hr. Avoid light from this step onwards.</li>
			<li>Counterstain nuclei with 4&#8242;, 6-diamidin-2-phenylindol (DAPI) diluted 1:5,000 in PBS at RT for 10 min.</li>
			<li>Wash 3 x 10 min with 0.5 ml PBS per chamber.</li>
			<li>Remove the chambers from the glass slide and dry. Mount with 250 &#181;, mounting medium per slide.</li>
		</ol>
		</li>
		<li>Proximity ligation assay
		<ol>
			<li>Prepare proximity ligation assay probes. Mix and dilute two proximity ligation assay probes (anti-mouse and anti-rabbit secondary antibodies attached with different oligonucleotides which can ligate through the addition of two other oligonucleotides in ligation solution) both 1:5 in 0.1% Triton X-100 and 5% NGS in PBS, with a total volume of 320 &#181;l for one 8-chamber slide (approximately 40 &#181;l per cm2). Incubate at RT for 20 min.</li>
			<li>Remove the chambers from the glass slide and add the diluted probes. Incubate in a humidity incubator at 37 &#176;C for 1 hr.</li>
			<li>Prepare ligation solution. Mix 8 &#181;l ligase, 64 &#181;l 5 x ligation stock and 248 &#181;l H2O.</li>
			<li>Tap off the liquid from the slide, and wash 2 x 5 min in 1x Wash Buffer A (provided with the kit).</li>
			<li>Add ligation solution and incubate in a humidity incubator at 37 &#176;C for 30 min.</li>
			<li>Prepare amplification solution. Mix 4 &#181;l polymerase, 64 &#181;l 5x amplification stock and 252 &#181;l H2O. Avoid light from this step onwards.</li>
			<li>Tap off liquid from the slide, and wash 2 x 2 min in 1x Wash Buffer A.</li>
			<li>Add the amplification solution and incubate in a dark humidity incubator at 37 &#176;C for 100 min.</li>
			<li>Tap off the liquid from the slide, and wash 2 x 10 min in 1x Wash Buffer B (provided with the kit). Wash for 1 min in 0.01x Wash Buffer B.</li>
			<li>Dry the slide and mount with 250 &#181;l of a commercial mounting medium (with DAPI) per slide.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Examine fluorescent signals under a microscope using a 10X eyepiece lens and 20X objective lens. Acquire images by a CCD camera.</li>
</ol>

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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. 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The authors have nothing to disclose.

There are several benefits as well as shortcomings for both methods. As a relatively novel technique, an obvious advantage of proximity ligation assay is the feasibility to elucidate protein-protein interactions at single-cell level instead of a batch of heterogeneous cells. Images with high magnitude and resolution (e.g. by confocal microscope) provide the possibility for quantification by counting single fluorescent spots. In contrast, the conventional combination of co-immunoprecipitation technique with Weste...

Nuclear factor 90 (NF90) is a multi-isoform protein with numerous functions including the response to viral infection, regulation of interleukin-2 post-transcription and regulation of miRNA biogenesis 1-3. RBM3 is an RNA-binding protein, involved in translation and miRNA biogenesis and can be induced by various stressors including hypothermia and hypoxia 4-6. Recently, we found NF90 and RBM3 in a protein complex 7. The interaction of NF90 and RBM3 is essential to modulate protein kinase RNA-like endoplasmic reticulum kinase (PERK) activity in unfolded protein response 7. Both NF90 and RBM3 are located predominantly in the nucleus but a small proportion of NF90 and RBM3 shuttle into the cytoplasm and bind there to each other for specific functions, e.g. to regulate PERK activity. Therefore, it is important to visualize the distribution of NF90-RBM3 interactions in the subcellular compartment, which may indicate their various roles in respective compartment.
Decades ago, yeast two hybrid (Y2H) was developed to detect the interaction between two proteins 8. However, due to artificial construction of fused proteins, false positive results have restricted the application of this method. For a long time, co-immunoprecipitation was the main technique to analyze protein-protein interactions, especially in endogeneous conditions 9. To analyze the co-immunoprecipitated protein complex, Western blot is the most convenient technique, while mass spectrometry is used when super sensitivity and accuracy are desired. In recent years, proximity ligation assay has been developed as a novel method to detect protein-protein interactions in both cells and tissues in situ&#160;10,11.
Here, we compared the most popular co-immunoprecipitation method and relatively novel proximity ligation assay method in capturing NF90-RBM3 interaction in subcellular fractions. We also discussed the advantages and limitations of both techniques.

<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.

Figure 1 demonstrates that NF90 and RBM3 are both nuclear proteins and only a small fraction is present in the cytoplasm. Notably, there are three different bands stained positive for RBM3. The smallest just below 20 kDa reflects the correct size of RBM3 (the predicted molecular weight of RBM3 is 17 kDa). The origin of the two other bands remains to be investigated. Co-immunoprecipitation experiments with RBM3 as the bait protein revealed that NF90-RBM3 interactions are p...

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.

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 ...

visualization-protein-protein-interaction-nuclear-cytoplasmic

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Biochemistry

12.7K Views.  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.

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

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

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.

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

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.

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

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.

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.

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

Developmental Biology

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.

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

Neuroscience

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.

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

Biology

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.

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.

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

Assessing Protein Interaction and Localization in DNA Damage

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.

Author Spotlight: Combining Proximity Ligand Assay with Gamma-H2AX Staining to Characterize Protein Interactions in DNA Damage Response

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

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

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