This research was supported by grants of the Deutsche Forschungsgemeinschaft GraKo1121, Hu1011/2-1 and SFB940 to S.H.

Note: Detailed information regarding buffers indicated in the protocol is available in Table 1.
1. Fixation of Protein-protein Interactions by Formaldehyde Cross-linking in Living Cells
<ol>
	<li>To begin prepare growth media, stock solutions and buffer P1
	<ol>
		<li>Prepare 500 ml medium: The medium should provide conditions that support the interaction between membrane bait protein and prey proteins. In addition, one experiment should be performed under conditions where no interaction is expected (e.g. without cross-linking). 
		Note: We compare protein-protein interactions in stressed and non-stressed bacteria. Therefore, we prepare 500 ml Luria Bertani (LB) medium (Table 1) that we complement with a stress-inducing agent (e.g. 0.5 M NaCl) in a 2 L Erlenmeyer flask. For control, we use normal LB medium with 0.17 M NaCl (pH 7.0).</li>
		<li>Prepare Tris-buffer (20 mM Tris-HCl; pH 8.0) and 0.1 M ethylenediaminetetraacetic acid (EDTA), pH 8.0 stock solution (Table 1).</li>
		<li>Prepare buffer P1. Dissolve sucrose to a final concentration of 0.5 M in Tris-buffer. Sterilize buffer P1 by filtration and store it at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Grow cells expressing the membrane bait protein with a C-terminal Strep-tag fusion from a vector. Induce the expression of membrane bait proteins for a sufficient time. 
	Note: We induce the expression of bacterial membrane bait proteins at early-log phase (A600 = 0.3) by addition of 250 &#181;l 1 M isopropyl-&#946;-D-thiogalactopyranoside (IPTG) to 500 ml medium (final concentration: 0.5 mM). To allow sufficient expression, we incubate the bacteria until the late-log phase (A600 = 1.2).</li>
	<li>Prepare for each culture two centrifuge beakers with a minimum volume of 300 ml each and place them on ice.</li>
	<li>Perform formaldehyde cross-linking. 
	CAUTION: Formaldehyde is highly toxic. We recommend working under a safety fume hood.
	<ol>
		<li>Transfer the culture vessel under a safety fume hood. Split each sample into two samples, one omitting cross-linking (- X) and one including the formaldehyde cross-linking (+ X). Add 4 ml 37% formaldehyde solution to 250 ml culture to reach a final concentration of 0.6%.</li>
	</ol>
	</li>
	<li>Transfer the culture vessels back and grow cells for further 20 min as before.</li>
	<li>Fill your cultures into the centrifuge tubes under a safety fume hood. Collect cells by centrifugation at 3,000 x g for 30 min.</li>
	<li>Discard supernatant by carefully pipetting it up under a safety fume hood. Collect toxic formaldehyde-containing supernatant and dispose of it properly.</li>
	<li>In order to reach optimal yield during membrane protein preparation, prepare spheroplasts first. Here, we provide a protocol for spheroplast formation of Gram-negative bacteria:
	<ol>
		<li>Prepare buffer P2 (Table 1) from lyophilized powder. Gently dissolve 2 mg lysozyme in 0.1 M EDTA, pH 8, in a 1.5 ml tube. Always prepare buffer P2 freshly for the day of the experiment.</li>
		<li>Prepare Tris-buffer with protease inhibitor (buffer P3). To 10 ml of Tris-buffer, add 0.1 ml 1 M phenylmethylsulfonylfluoride (PMSF) (Table 1) to a final concentration of 10 mM. Use the buffer immediately, to ensure proper function of PMSF as protease inhibitor.</li>
		<li>Resuspend cell pellets in 10 ml Tris-buffer with protease inhibitor and transfer solution to a 15 ml conical tube.</li>
		<li>Add 1 ml buffer P2 and incubate on ice for 30 min.</li>
		<li>Collect spheroplasts by centrifugation at 3,000 x g for 30 min and discard supernatant carefully.</li>
	</ol>
	</li>
	<li>Incubate the spheroplast pellets overnight at -20&#176;C.</li>
</ol>
2. Purification of Strep-tag Membrane Protein (Bait)
<ol>
	<li>Place spheroplast pellet on ice.</li>
	<li>During the time the spheroplast pellets thaw on ice, prepare 10 ml buffer P3 for each spheroplast preparation. Gently dissolve 1 mg DNaseI in 10 ml Tris-buffer. Add 0.1 ml 1 M PMSF just before use.</li>
	<li>Resuspend the pellet in 6 ml freshly prepared P3. Sonicate the sample four times for 1 min continuously on ice with 1 min pause between each burst, in order to disrupt spheroplasts.</li>
	<li>Centrifuge the sample at 10,000 x g for 10 min to harvest cell debris. Transfer the supernatant using a pipette to an ultracentrifuge tube.</li>
	<li>Pellet the membrane fraction at 100,000 x g for 30 min. Wash the pellet carefully in Tris-buffer without dissolving it. Dry the tube with a paper tissue (e.g. Kleenex). Avoid disturbing the pellet at any time.</li>
	<li>Resuspend the pellet (= membranes) carefully in 1 ml Tris-buffer. Use a 20 &#181;l aliquot to determine the protein concentration by e.g. BCA assay. At this step, the membranes can be shock frozen in liquid nitrogen and stored at -80 &#176;C.</li>
</ol>
3. Purification of Strep-tag Membrane Bait Protein and SDS-PAGE
<ol>
	<li>Normalize the protein concentration of the membrane fraction to 5 mg/ml with Tris-buffer. Take 2.5 ml membrane fraction (5 mg/ml) in an ultracentrifuge tube and add 0.25 ml of 20% Triton X-100 to reach a final concentration of 2%, in order to solubilize the membrane proteins. Add a micro-magnetic rod and stir on ice for 1 hr. 
	Note: Although in general we have good results using Triton X-100 as detergent, it might be useful to change the detergent for optimization. In this respect, we have best results with those detergents used for functional purification of the respective membrane bait protein.</li>
	<li>While performing step 3.1, prepare 50 ml buffer W and 5 ml buffer E from (Table 1). Fill up 10 ml 5x buffer W concentrate to 50 ml and add 150 &#181;l 20% Triton X-100.
	<ol>
		<li>Fill up 1 ml 5x buffer E concentrate to 10 ml and add 30 &#181;l 20% Triton X-100. Equilibrate a 1 ml Strep-Tactin superflow gravity flow column with 8 ml buffer W. 
		Note: It is essential&#160;to add the detergent used for solubilization in a concentration 10-fold above the critical micelle concentration (cmc) in any buffer during the purification procedure.</li>
	</ol>
	</li>
	<li>Remove the micro-magnetic rod from the solubilization sample and ultracentrifuge at 100,000 x g for 30 min, to pellet the insoluble membrane fraction. Remove the supernatant using a pipette for further purification.</li>
	<li>Load the supernatant to the column. Run the column only with gravity flow. Wash the column with 5 ml buffer W. Repeat this washing step 5x.</li>
	<li>Elute membrane bait proteins with 1 ml buffer E. Repeat this elution step 4x.</li>
	<li>Concentrate elution fractions 2, 3, and 4 to 300 &#181;l with a centrifugal filter unit.</li>
	<li>Mix 200 &#181;l of each sample with 50 &#181;l 5x SDS-PAGE loading dye. Split each preparation in 125 &#181;l aliquots. Boil one aliquot of each preparation for 20 min at 95 &#176;C, to reverse formaldehyde cross-links. Let samples cool down to RT for at least 10 min on bench top.</li>
	<li>Load 30 &#181;l from each sample to a single lane of a polyacrylamide-gel suitable for immunoblotting. Use a prestained molecular weight marker for best orientation and run SDS-PAGE10.</li>
</ol>
4. Immunoblot Analysis to Confirm Interaction Partners
<ol>
	<li>Once step 3.8 is completed, transfer proteins to a nitrocellulose membrane by e.g. semidry.</li>
	<li>Block the membrane to prevent unspecific labeling. Prepare blocking buffer by weighing bovine serum albumin (BSA) to achieve 3% weight per volume in Tris-buffered saline supplemented with final a concentration of 0.05% Tween-20 (TBS-T). Use a sufficient volume of blocking buffer to cover the membrane. Block the membrane for 1 hr at RT.</li>
	<li>Dilute and incubate the prey protein specific first antibody as usual. Use an HRP-linked antibody as the second antibody.</li>
	<li>Develop the immunoblot using a chemiluminescent detection kit with high sensitivity. Monitor the signal using a classical film processing procedure or digital imaging equipment.</li>
</ol>
5. NanoLC-ESI-MS/MS High Resolution Experiments to Identify Interaction Partners
<ol>
	<li>In case that no specific antibody is available for the prey protein or unknown interaction partners should be identified, use mass spectrometry (MS) for identification. In order to prevent keratin contamination, use precasted gels for protein separation.</li>
	<li>Silver stain the SDS-PAGE using a MS-compatible staining kit according to the manufacturer&#39;s protocol. Perform all staining and washing steps in glass tanks.</li>
	<li>Excise respective bands and analyze these by high resolution LC/MS9.</li>
</ol>

<ol>
	<li>Krogh, A., Larsson, B., von Heijne, G., Sonnhammer, E. L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+transmembrane+protein+topology+with+a+hidden+markov+model:+application+to+complete+genomes.">Predicting transmembrane protein topology with a hidden markov model: application to complete genomes.</a> J. Mol. Biol. 305, 567-580 (2001).</li><li>Bakheet, T. M., Doig, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+and+identification+of+human+protein+drug+targets.">Properties and identification of human protein drug targets.</a> Bioinformatics. 25, 451-457 (2009).</li><li>Yildirim, M. A., Goh, K. I., Cusick, M. E., Barabasi, A. L., Vidal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-target+network.">Drug-target network.</a> Nat. Biotechnol. 25, (2007).</li><li>Daley, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+assembly+of+membrane+proteins+into+complexes.">The assembly of membrane proteins into complexes.</a> Curr. Opin. Struct. Biol. 18, 420-424 (2008).</li><li>Jung, K., Fried, L., Behr, S., Heermann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histidine+kinases+and+response+regulators+in+networks.">Histidine kinases and response regulators in networks.</a> Curr. Opin. Microbiol. 15, 118-124 (2012).</li><li>M&#252;ller, V. S., Jungblut, P. R., Meyer, T. F., Hunke, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-SPINE:+An+improved+method+to+identify+protein-protein+interaction+partners+of+membrane+proteins+in+vivo.">Membrane-SPINE: An improved method to identify protein-protein interaction partners of membrane proteins in vivo.</a> Proteomics. 11, 2124-2128 (2011).</li><li>Herzberg, C., 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=SPINE:+A+method+for+the+rapid+detection+and+analysis+of+protein-protein+interactions+in+vivo.">SPINE: A method for the rapid detection and analysis of protein-protein interactions in vivo.</a> Proteomics. 7, 4032-4035 (2007).</li><li>Sutherland, B. W., Toews, J., Kast, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+formaldehyde+cross-linking+and+mass+spectrometry+in+the+study+of+protein-protein+interactions.">Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions.</a> J. Mass Spectrom. 43, 699-715 (2008).</li><li>Hernandez, P., M&#252;ller, M., Appel, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+protein+identification+by+tandem+mass+spectrometry:+Issues+and+strategies.">Automated protein identification by tandem mass spectrometry: Issues and strategies.</a> Mass Spectrom. Rev. 25, 235-254 (2006).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Hunke, S., Keller, R., M&#252;ller, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+integration+by+the+Cpx-envelope+stress+system.">Signal integration by the Cpx-envelope stress system.</a> FEMS Microbiol. Lett. 326, 12-22 (2012).</li><li>Weber, R. F., Silverman, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cpx+proteins+of+Escherichia+coli+K12+:+Structure+of+the+CpxA+polypeptide+as+an+inner+membrane+component.">The Cpx proteins of Escherichia coli K12 : Structure of the CpxA polypeptide as an inner membrane component.</a> J. Mol. Biol. 203, 467-478 (1988).</li></ol>

We have nothing to disclose.

Membrane SPINE analysis is a biochemical approach that enables one to confirm and to identify to this point unknown interaction partners of membrane proteins. Membrane SPINE combines in vivo cross-linking by formaldehyde with purification of a Strep-tagged membrane bait protein. The combination with immunoblotting facilitates the confirmation of predicted interaction partners (Figure 2). Additionally, the combination with MS analysis permits the identification of unknown interaction partners (<s...

To understand the function of a protein it is essential to know its interaction partners. Several classical techniques are available for the identification of interaction partners of soluble proteins. However, these techniques are not easily transferable to membrane proteins due to their hydrophobic nature4. To overcome this limitation, we have developed the Membrane Strep-protein interaction experiment (Membrane-SPINE)6. It is based on the SPINE method, which was only suitable for soluble proteins7.
Membrane-SPINE benefits from two advantages of the cross-linking agent formaldehyde: first, formaldehyde can easily penetrate membranes and therefore generates a precise snapshot of the interactome of a living cell8. Second, formaldehyde cross-links can be reversed by boiling9. Here, these two advantages are used to identify not only permanent but also transient PPIs of membrane proteins6.
In brief, a Strep-tag is fused to the C-terminus of the integral membrane bait protein. Cells expressing the membrane bait protein are incubated with formaldehyde which cross-links prey proteins to the membrane bait protein (Figure 1). Modifications of prey proteins are not needed. Next, the membrane fraction is prepared. Therefore, membrane proteins are solubilized by detergent treatment and bait proteins are co-purified with its prey proteins using affinity purification. Subsequently, the cross-link is reversed by boiling, and the bait and its co-eluted prey proteins are separated by SDS-PAGE. Finally, prey proteins can be identified by immunoblot analysis or mass spectrometry.

<table><tbody><tr><td>37% Formaldehyde</td><td>Roth</td><td>4979.1</td><td>Should not be older than one year</td></tr><tr><td>DNaseI</td><td>Sigma</td><td>DN25</td><td></td></tr><tr><td>BCA protein assay kit</td><td>Pierce</td><td>23225</td><td></td></tr><tr><td>Micromagnetic rod</td><td>Roth</td><td>0955.2</td><td>5 mm in length, 2 mm in diameter</td></tr><tr><td>Triton X-100</td><td>Roth</td><td>6683.1</td><td>standard detergent for solubilization</td></tr><tr><td>n-Dodecyl-&amp;beta;-maltoside</td><td>Glycon</td><td>D97002-C</td><td>the best detergent to solubilze CpxA</td></tr><tr><td>1 ml Strep-Tactin superflow gravity flow column</td><td>IBA</td><td>2-1207-050</td><td></td></tr><tr><td>Strep-tag protein purification buffer set</td><td>IBA</td><td>2-1002-001</td><td>Contains buffer W and buffer E</td></tr><tr><td>Amicon Ultra-4 centrifugal filter</td><td>Millipore</td><td>UFC803024</td><td></td></tr><tr><td>SuperSignal West Pico Chemiluminescent substrate</td><td>Pierce</td><td>34080</td><td></td></tr><tr><td>SilverQuest Silverstaining kit</td><td>Invitrogen</td><td>LC6070</td><td></td></tr><tr><td>FireSilver Staining Kit</td><td>Proteome Factory</td><td>P-S-2001</td><td></td></tr><tr><td>Ultracentrifuge</td><td></td><td></td><td></td></tr></tbody></table>

membrane-spine: a biochemical tool to identify protein-protein interactions of membrane proteins in vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane SPINE analysis allows the co-purification of membrane proteins and transiently interacting protein partners. The co-purification is achieved by using the cross-linking agent formaldehyde. Two parameters are critical to prevent unspecific cross-links: the formaldehyde concentration and the cross-linking time. The sufficient, but not excessive use of formaldehyde can easily be controlled by immunoblotting. Formaldehyde cross-linked protein complexes can be separated by boiling but not by SDS treatment. Hence, they...

Watch this Scientific Journal Video about Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo at JoVE.com

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

membrane-spine-biochemical-tool-to-identify-protein-protein

Research

JoVE Journal

Bioengineering

13.7K Views.  Universität Osnabrück. A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

Video: Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Biochemistry

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

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 ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Biology

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, ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Biotinylated Cell-Penetrating Peptide Probe to Detect Protein-Protein Interactions

Source: Jara&#237;z-Rodr&#237;guez, M. et al., <a href="https://app.jove.com/v/56457">Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions.</a> J. Vis. Exp. (2017).
This video demonstrates a technique that uses biotinylated cell-penetrating peptides to detect intracellular protein-protein interactions. The cell-penetrating peptides facilitate the conjugated peptide to enter a cell and specifically bind to its interacting proteins. The biotin, after binding to avidin, allows purification of the interacting proteins&#8217; complex from a cell lysate.

Biotynylowana sonda peptydowa penetrująca komórki do wykrywania interakcji białko-białko

Source: Jara&#237;z-Rodr&#237;guez, M. et al., Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions. J. Vis. Exp. ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

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Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Biotynylowana sonda peptydowa penetrująca komórki do wykrywania interakcji białko-białko