We thank the entire Chernoff laboratory for contributing to the optimization and validation of this protocol, in particular Maria Radu and Galina Semenova. We also thank Andrey Efimov of the Cell Imaging Facility at Fox Chase Cancer Center. This work was supported by a grant from the NIH (R01 CA148805) to JC.

1. Preparation of Solutions
<ol>
	<li>Prepare fixative solution: 4% paraformaldehyde (PFA) in 1x PBS. For 10 mL, take 2.5 mL of 16% PFA and add 7.5 mL of 1x PBS. 
	Hazards: PFA is carcinogenic at low doses. Fumes and skin contact are hazardous. Store at -20 &#176;C.</li>
	<li>Prepare permeabilization solution: 0.1% Triton X-100 in 1x PBS. For 100 mL of solution, add 100 &#181;L of Triton X-100 into 100 mL of 1x PBS. Store at room temperature (RT).</li>
	<li>Prepare Wash Buffer: 1x TBST. For 1 L, take 100 mL of 10x TBS, 890 mL of dH2O, and 10 mL of Tween 20 (10%).</li>
	<li>Prepare blocking solution as supplied by kit. Alternatively, use 1x PBS solution containing 2% BSA.</li>
	<li>Prepare the antibody diluent as supplied by kit. Alternatively, use 1x PBS solution containing 1% BSA.</li>
</ol>
2. Plating of Cells
NOTE: In this study, we used immortalized Human Schwann Cells (iHSC) and Human Embryonic Kidney 293 cells (HEK 293); however, this method can be used for many types of adherent cells.
<ol>
	<li>Culture HEK 293 in DMEM supplemented with 10% FBS, 0.2% Glucose, 2 mM L-Glutamine, 100 U/mL penicillin G and 100 &#181;g/mL streptomycin on tissue culture-treated plates at 37 &#176;C in 5% CO2. Maintain iHSC in 10% FBS/DMEM supplemented with Pen/Strep and 2 &#181;M forskolin. Routinely test cells for mycoplasma. No cell line authentication was performed.</li>
	<li>Coat a 16-well chamber slide with 50 &#181;L of 10 &#181;g/mL natural mouse laminin or 0.01% poly-L-lysine solution and incubate for 30 min at 37 &#176;C in 5% CO2. Split cells using 0.04% trypsin-EDTA.</li>
	<li>Plate iHSC and HEK-293 in 100 &#181;L of medium at 80% confluence (15,000-25,000 cells/well).</li>
	<li>Incubate the cells for 12 -24 hours at 37 &#176;C in a humidified, 5% CO2 incubator.</li>
</ol>
3. Fixation and Permeabilization
<ol>
	<li>Remove the medium from the wells and wash with 100 &#181;L of 1x PBS. Aspirate with a micropipette to minimize the risk of removing the sample.</li>
	<li>Fix cells by adding 50 &#181;L of 4% PFA per well and incubate for 10 min at RT, without agitation. Cells are sensitive to detachment, so avoid pipetting the solutions directly onto the cells.</li>
	<li>Wash the cells with 0.05% TBST three times for 5 min each. Aspirate with a micropipette to minimize the risk of removing the sample.</li>
	<li>Treat the cells with permeabilization solution (0.1% Triton x-100 in 1x PBS) for 10 min without agitation at RT.</li>
	<li>Wash cells with TBST three times for 5 min each with agitation.</li>
</ol>
4. Blocking
<ol>
	<li>Tap off the TBST (very gently with a micropipette). Visualize the slide in a microscope to see if cells remain attached.</li>
	<li>Add one drop (50-60 &#181;L) of 1x Blocking Solution to each sample.</li>
	<li>Pre-heat a humidity chamber (empty tip box with water) at 37 &#176;C and incubate the slides in it for 1 h at 37 &#176;C.</li>
</ol>
5. Primary Antibodies
<ol>
	<li>Dilute the primary antibodies (1:100) in the Antibody Diluent (see Table of Materials). Prepare 40 &#181;L antibody solution per well.</li>
	<li>Remove blocking solution from the slides by tapping off the liquid. Do not allow cells to dry.</li>
	<li>Vortex and add 40 &#181;L of the antibody solutions to each well.</li>
	<li>Incubate 1 h at 37 &#176;C in a preheated humidity chamber.</li>
</ol>
6. Proximity Ligation Assay Probes
NOTE: PLA probes are provided as part of a kit (see Table of Materials). The choice of probes will depend on the species of primary antibodies used to detect the proteins of interest.
<ol>
	<li>Dilute the two PLA probes (anti-Mouse MINUS and anti-Rabbit PLUS) 1:5 in the Antibody Diluent. For each sample, prepare 40 &#181;L of PLA probe. Incubate the mixture for 20 min at RT.</li>
	<li>Tap off the primary antibody solution from the slides. Wash the slides twice for 5 min each with Wash Buffer at RT.</li>
	<li>Gently tap of the Wash Buffer from the slides and add the diluent PLA probe solution to wells (40 &#181;L/well).</li>
	<li>Incubate the slides in a pre-heated humidity chamber for 1 h at 37 &#176;C.</li>
</ol>
7. Ligation
NOTE: The in situ Detection Reagents Red, Ligase, and Ligation Solution are provided as part of a kit (see Table of Materials).
<ol>
	<li>Use the in situ Detection Reagents Red.</li>
	<li>Immediately before use, vortex and dilute the required volumes of the 5x ligation stock 1:5 in high purity water. 
	NOTE: Do not store diluted reagents.</li>
	<li>Tap off the PLA probe solution from the slides. Wash the slides in 1x Wash Buffer twice for 5 min each at RT.</li>
	<li>Immediately before addition to the samples, vortex and add Ligase to the Ligation solution at 1:40 dilution and vortex again.</li>
	<li>Tap off the Wash Buffer from the slides and add the Ligation/Ligase solution to each well (40 &#181;L/well).</li>
	<li>Incubate the slides in a pre-heated humidity chamber for 30 min at 37 &#176;C.</li>
</ol>
8. Amplification
NOTE: The amplification stock is provided as part of a kit (see Table of Materials). Reagents are light sensitive; thus avoid exposing the slides to light.
<ol>
	<li>Vortex and dilute the required volumes of the 5x Amplification stock 1:5 in high purity water and mix.</li>
	<li>Tap off the Ligation/Ligase solution from the slides. Wash the slides in 1x Wash Buffer twice for 2 min each at RT.</li>
	<li>Vortex and add the polymerase to the Amplification solution at 1:80 dilution and vortex again.</li>
	<li>Tap off Wash Buffer from the slides and add the Amplification/Polymerase solution to each well (40 &#181;L/well). Incubate the slides in a pre-heated humidity chamber for 100 min at 37 &#176;C.</li>
</ol>
9. Preparation for Imaging
NOTE: These are light sensitive reagents. Keep the slides protected from light.
<ol>
	<li>Tap off the Amplification-Polymerase solution from the slides and wash twice for 10 min each in 1x Wash Buffer at RT.</li>
	<li>Remove the chambers and silicone around the wells from the slide completely. Scrape off the remaining bits of silicone with a razor. Draw a grid on the slide separating each well.</li>
	<li>Add ~40 &#181;L of the mounting medium with DAPI. Be careful to avoid trapping air bubbles under the cover slip. The edges of the cover slip can be sealed with nail polish.</li>
	<li>Wait for at least 20 min before performing image analysis using a confocal microscope. 
	NOTE: The protocol can be paused here.</li>
	<li>After imaging, store the slides at -20 &#176;C in the dark.</li>
</ol>

<ol>
	<li>Zhou, D., 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+Nore1B/Mst1+complex+restrains+antigen+receptor-induced+proliferation+of+na&#239;ve+T+cells.">The Nore1B/Mst1 complex restrains antigen receptor-induced proliferation of na&#239;ve T cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (51), 20321-20326 (2008).</li><li>Praskova, M., Xia, F., Avruch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MOBKL1A/MOBKL1B+phosphorylation+by+MST1+and+MST2+inhibits+cell+proliferation.">MOBKL1A/MOBKL1B phosphorylation by MST1 and MST2 inhibits cell proliferation.</a> Current Biology. 18 (5), 311-321 (2008).</li><li>Dan, I., Watanabe, N. M., Kusumi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ste20+group+kinases+as+regulators+of+MAP+kinase+cascades.">The Ste20 group kinases as regulators of MAP kinase cascades.</a> Trends in Cell Biology. 11 (5), 220-230 (2001).</li><li>Rawat, S. 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=H-ras+Inhibits+the+Hippo+Pathway+by+Promoting+Mst1/Mst2Heterodimerization.">H-ras Inhibits the Hippo Pathway by Promoting Mst1/Mst2Heterodimerization.</a> Current Biology. 26 (12), 1556-1563 (2016).</li><li>Creasy, C. L., Ambrose, D. M., Chernoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ste20-like+protein+kinase,+Mst1,+dimerizes+and+contains+an+inhibitory+domain.">The Ste20-like protein kinase, Mst1, dimerizes and contains an inhibitory domain.</a> Journal of Biological Chemistry. 271 (35), 21049-21053 (1996).</li><li>Buntru, A., Trepte, P., Klockmeier, K., Schnoegl, S., Wanker, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Approaches+Toward+Quantitative+Mapping+of+the+Interactome.">Current Approaches Toward Quantitative Mapping of the Interactome.</a> Frontiers in Genetics. 74 (7), (2016).</li><li>Greenwood, C., Ruff, D., Kirvell, S., Johnson, G., Dhillon, H. S., Bustin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+assays+for+sensitive+quantification+of+proteins.">Proximity assays for sensitive quantification of proteins.</a> Biomolecular Detection and Quantification. 4, 10-16 (2015).</li><li>Fredriksson, 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=Protein+detection+using+proximity-dependent+DNA+ligation+assays.">Protein detection using proximity-dependent DNA ligation assays.</a> Nature Biotechnology. 20 (5), 473-477 (2002).</li><li>Leuchowius, K. 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=Parallel+visualization+of+multiple+protein+complexes+in+individual+cells+in+tumor+tissue.">Parallel visualization of multiple protein complexes in individual cells in tumor tissue.</a> Molecular &amp; Cellular Proteomics. 12 (6), 1563-1571 (2013).</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> Nature Methods. 3 (12), 995-1000 (2006).</li></ol>

The authors declare that they have no competing financial interests.

We found it useful to use glass chamber slides for this experiment, as it is very convenient to perform experiment with several (14-16) cell lines and there is no need to change the sample every time during microscopy analysis. A few complications may arise, such as an increased risk for cross-contamination with antibodies. Therefore, we suggest washing every well individually instead of using a Coplin jar, despite the increased duration of the experiment. In addition, removal of silicone insert is a delicate affair and ...

Disruption of MST1/Hippo signaling has been connected to developmental disorders and carcinogenesis1. In mammals, the kinases MST1 and MST2 activate (phosphorylate) MOB1 and LATS1/2, the latter of which then phosphorylates and inactivates the transcriptional co-activator Yes-associated protein (YAP)2. In its active (unphosphorylated) form, YAP has oncogenic activity, enhancing transcription of cell proliferation genes; conversely, when YAP is inactivated by the Hippo pathway, cell proliferation is suppressed and apoptosis promoted3. In tissues, MST1 and MST2 exist mainly as active homodimers, but oncogenic stimuli can increase levels of MST1/MST2 heterodimers, and such heterodimers are inactive4. However, how MST1/MST2 heterodimerization is regulated remains poorly understood. Both homo- and heterodimers are mediated via interactions between C-terminal coiled-coil regions of MST1 and MST2 known as SARAH domains5. Using an in situ PLA demonstrated in this article, we show the presence of MST1/MST2 heterodimers in Human Schwann Cells (HSC) and Human Embryonic Kidney cells (HEK-293). PLA has an advantage over other protein/protein interaction detection methods because it allows the detection of endogenous protein-protein interactions, which can be identified and quantified without the need of transgene expression or the use of epitope tags6.
Signaling transduction pathways are largely controlled by the conditional association of component proteins. For example, stimulation of most receptor tyrosine kinases leads to their homo- or hetero-dimerization and subsequent association with additional intracellular signaling proteins, which themselves form further complexes. The purpose of the PLA method is to visualize proximity between the proteins in cells, provided that the proteins are less than 30-40 nm apart. Protein proximity is usually detected by first incubating the cells with appropriate primary antibodies raised in different species (e.g., rabbit and mouse) against each interacting protein, then adding species-specific secondary antibodies, pre-coupled to short DNA probes. If the DNA probes are in close proximity, a specific linking DNA oligonucleotide can simultaneously bind both of these probes, forming a platform for amplification by in situ PCR or by rolling circle mechanism. Fluorescent tags added to the amplification reaction allow visualization of the interacting proteins, which appear as fluorescent dots that can be readily quantified and localized to particular regions in the cell7,8,9,10.

<table border="0" cellpadding="0" cellspacing="0" style="width:849px;" width="849">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Chamber slides</td>
			<td style="width:123px;">Thermo Fisher Scientific</td>
			<td style="width:344px;">178599</td>
			<td style="width:167px;">16 well, glass slide</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PLA probe Anti-Mouse Minus</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92004</td>
			<td>Contains 1x Blocking solution, 1x antibody Diluent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PLA Probe Anti-Rabbit PLUS</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92002</td>
			<td>Contains 1x Blocking solution, 1x antibody Diluent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Wash Buffers, Flurescence</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82049</td>
			<td>Contains Wash Buffer A and Wash Buffer B</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mounting Medium with DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82040</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Detection Reagents Red</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92008</td>
			<td>Contains 5x Ligation, 1x Ligase, 5x Amlification Red, 1x Polymerase</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">p44/42 MAPK (Erk1/2) Antibody</td>
			<td>Cell Signaling</td>
			<td>9102s</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Phospho-p44/42 MAPK (Erk1) (Tyr204)/(Erk2) (Tyr187)</td>
			<td>Cell Signaling</td>
			<td>5726s</td>
			<td>pERK antibody</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MST2 antibody</td>
			<td>Cell Signaling</td>
			<td>3952s</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Krs-2 (RJ-5)</td>
			<td>Santa Cruz</td>
			<td>sc-100449</td>
			<td>MST1 antibody</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">16% Paraformaldehyde</td>
			<td>Electron microscopy sciences</td>
			<td>15710</td>
			<td>Dilute to 4% PFA in PBS for fixing solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Triton x100</td>
			<td>Fisher BioReagents</td>
			<td>BP 151-500</td>
			<td>To prepare 0.1% Triton x-100 in 1xPBS for Permeabilization solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Confocal microscopy</td>
			<td>Leica TCS SP8, 63x</td>
			<td></td>
			<td>Image analysis with ImageJ software</td>
		</tr>
	</tbody>
</table>

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.

We used the PLA assay to test the interaction between MST1 and MST2 in HEK-293 and iHSC. The cells were fixed, permeabilized, and stained with various antibodies, followed by in situ amplification according to the PLA protocol (Figure 1). To document the level of MST1/MST2 heterodimerization, cells were stained with MST1 and MST2 antibodies (Figure 1A, 1C, and 1G). As a positive control, we also used ERK...

Watch this Scientific Journal Video about Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay at JoVE.com

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.

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 ...

detection-heterodimerization-protein-isoforms-using-an-situ-proximity

Research

JoVE Journal

Biochemistry

7.4K Views.  Fox Chase Cancer Center. Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

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

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules. Besides, the electro-driven separation in CZE is governed by extensive properties of matter and offers the advantage of analyzing proteins close to their native state. However, the successful implementation of this technique for routine analysis depends on the skills of the analyst at the critical steps during sample and system preparation. The purpose of this tutorial is to detail the steps to succeed in the CZE analysis of mAbs. Further, this protocol can be used for the development and improvement of skills of the personnel involved in protein analytical chemistry laboratories.

Here, we present a comprehensive capillary zone electrophoresis protocol for the assessment of intrinsic physicochemical heterogeneity of monoclonal antibodies as a quality attribute.

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The ...

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.

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.

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

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 ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...