The authors acknowledge NSERC RGPIN-2018-04994, Campus Alberta Innovation Program (RCP-12-002C), and Alberta Prion Research Institute/Alberta Innovates Bio Solutions (201600018) grants awarded to M.O.&#160;

<ol>
	<li>Jin, L., Wang, W., Fang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+protein-protein+interaction+by+small+molecules.">Targeting protein-protein interaction by small molecules.</a> Annual Review of Pharmacology and Toxicology. 54, 435-456 (2014).</li><li>Titeca, K., Lemmens, I., Tavernier, J., Eyckerman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovering+cellular+protein-protein+interactions:+Technological+strategies+and+opportunities.">Discovering cellular protein-protein interactions: Technological strategies and opportunities.</a> Mass Spectrometry Reviews. 38 (1), 79-111 (2019).</li><li>Cui, H., Loftus, K. M., Noell, C. R., Solmaz, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Cyclin-dependent+Kinase+1+Specific+Phosphorylation+Sites+by+an+In+Vitro+Kinase+Assay.">Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay.</a> Journal of Visualized Experiments. (138), e57836 (2018).</li><li>Zheng, X., 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=Co-immunoprecipitation+Assay+Using+Endogenous+Nuclear+Proteins+from+Cells+Cultured+Under+Hypoxic+Conditions.">Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions.</a> Journal of Visualized Experiments. (138), e57836 (2018).</li><li>Nolan, R., 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=Calibration-free+Vitro+of+Protein+Homo-oligomerization+Using+Commercial+Instrumentation+and+Free,+Open+Source+Brightness+Analysis+Software.">Calibration-free Vitro of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software.</a> Journal of Visualized Experiments. (137), e58157 (2018).</li><li>Luzarowski, M., Wojciechowska, I., Skirycz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2+in+1:+One-step+Affinity+Purification+for+the+Parallel+Analysis+of+Protein-Protein+and+Protein-Metabolite+Complexes.">2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes.</a> Journal of Visualized Experiments. (138), e57720 (2018).</li><li>Karchugina, S., 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=Detection+of+Heterodimerization+of+Protein+Isoforms+Using+an+Situ+Ligation+Assay.">Detection of Heterodimerization of Protein Isoforms Using an Situ Ligation Assay.</a> Journal of Visualized Experiments. (140), e57755 (2018).</li><li>Winkelaar, G., Trieber, C., Kumar, J., Overduin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Interactions+of+Globular+and+Filamentous+Proteins+by+Nuclear+Magnetic+Resonance+Spectroscopy+(NMR)+and+Microscale+Thermophoresis+(MST).">Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST).</a> Journal of Visualized Experiments. (141), e58537 (2018).</li><li>Mrozowich, T., McLennan, S., Overduin, M., Patel, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Studies+of+Macromolecules+in+Solution+using+Small+Angle+X-Ray+Scattering.">Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering.</a> Journal of Visualized Experiments. (141), e58538 (2018).</li><li>Stumpf, M. P., 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=Estimating+the+size+of+the+human+interactome.">Estimating the size of the human interactome.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (19), 6959-6964 (2008).</li><li>Lundberg, E., Borner, G. H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+proteomics:+a+powerful+discovery+tool+for+cell+biology.">Spatial proteomics: a powerful discovery tool for cell biology.</a> Nature Reviews Molecular Cell Biology. , (2019).</li></ol>

The authors have nothing to disclose.

Protein-protein interactions (PPIs) are fundamental to the generation of biological effects. Many are intimately linked with disease, and they are increasingly recognized as offering important targets for drug action1. Numerous techniques are available to study these protein interactions, some of which are discussed in this collection or in other JoVE articles. Each technique has its own merits and limitations2; however, there is no one perfect technique for all PPIs due to the massive diversity of these interactions which occur in different subcellular compartments, with a wide range of affinities (picomolar to millimolar), and may be only transient in nature.
In vitro kinase assays are an indirect approach for studying PPIs that can be used to analyze how kinases perform their functional role of transferring a phosphate group from ATP to a substrate. Cui et al.3 use this technique, coupled with tryptic digest and mass spectrometry, to identify the cyclin-dependent kinase-1-specific phosphorylation sites on a fragment of the human centromere protein F (CENP-F). For functional verification of novel phosphorylation sites, the authors use a binding assay, combining CENP-F containing a phosphomimetic mutation and karyopherin &#945;, a nuclear transport receptor, thus providing cross-validation.
Coimmunoprecipitation is a widely used method for studying PPIs in which an antibody is used to capture a specific target together with any other protein molecules that are associated with it. Zheng and colleagues4 apply this technique to study hypoxia-inducible factors (HIFs) that function as heterodimers composed of an oxygen-regulated &#945; subunit and a constitutively expressed &#946; subunit also known as ARNT3. An interesting note about their protocol is that the cells are induced and harvested under hypoxic conditions, and endogenous proteins from nuclear fractions are then coimmunoprecipitated. As the authors note, one limitation of the method is that it cannot determine if PPIs are direct or indirect.
Nolan et al.5 present a protocol for the measurement of the number of molecules and their brightness in fluorescence microscopy images &#8216;Number and Brightness&#8217; (N&#38;B), that can be applied to detecting protein homo-oligomerization. The technique can be used both in vitro and in vivo and has been applied to accurately quantitate the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of a dimerizing drug. The authors discuss the merits of the technique relative to related technologies and highlight the importance of correcting for bleaching and long-term intensity fluctuations. Confocal microscopes equipped with digital detectors make quantitation easier, but N&#38;B is also possible with analog detectors.
Affinity purification of multiprotein complexes, together with the identification of their components, has been a widely used technique for understanding the functions of such complexed proteins. In the protocol by Luzarowski et al.6, this technique is taken a step further by simultaneously characterizing both PPIs and protein-metabolite interactions (PMIs) in transgenic Arabidopsis plant cells. Tagged nucleoside diphosphate kinases are affinity purified by the tandem-affinity purification (TAP) tag procedure with captured proteins and small molecules subsequently being identified by mass spectrometry. The uniqueness of this protocol is the ability to identify both hydrophobic and hydrophilic ligands; it is a three-in-one extraction protocol that enables both PPIs and PMIs to be studied and applies affinity purification to the planting of cells.
The proximity ligation assay has the advantage of being used to detect interactions between endogenous proteins in cells and tissues. Karchugina and Chernoff7 use the technique to show the presence of heterodimers of the kinases MST1 and MST2 in human Schwann cells (HSCs) and human embryonic kidney cells (HEK-293). Their article has several valuable pointers on how to perform the technique successfully. Appropriate primary antibodies raised in different species against each interacting protein are required, and since the specificity and sensitivity of the antibodies are key, antibody concentrations should be finely tuned. The authors found glass chamber slides convenient for analyzing multiple cell lines and antibody combinations but noted that it is imperative to remove all of the silicone insert between wells to avoid differences in the confocal distance during microscopy. In addition, various positive and negative controls are used to validate the results.
Nuclear magnetic resonance (NMR) spectroscopy is a sensitive assay that can be used to provide atomic-level resolution and quantitative information about protein interactions and protein-ligand interactions. Winkelaar and colleagues8 use the technique to study the interaction of two proteins (vimentin and envoplakin) found in desmosomes, cell structures involved in cell-to-cell adhesion that are typically found in tissues that experience intense mechanical stress. The authors use a 15N-labelled vimentin domain and acquire a 2D spectrum using heteronuclear single quantum correlation (HSQC). In the presence of a domain from envoplakin, extensive line broadening and peak disappearance are observed, consistent with protein interaction. The authors further probe the basis of the interaction using mutagenesis and microscale thermophoresis (MST) to provide quantitative information.
Small-angle X-ray scattering (SAXS) is a technique that can be used to provide information on the size and conformations of a macromolecule in solution and generate a molecular envelope of large, multidomain or protein complexes at low resolution. In the protocol by Mrozowich et al.9, the technique is used to generate a low-resolution structure of nidogen-1 and laminin &#947;-1 in complex. The technique may be of interest to researchers studying PPIs in large proteins with multiple globular domains that are too big for NMR spectroscopy and are challenging to crystallize for X-ray crystallography.
It has been estimated there are some 650,000 interactions in the human interactome10&#8212;enough to keep researchers in the field busy for some time to come. A recent development, made possible by advances in microscopy, mass spectrometry, and machine learning, is spatial proteomics that may provide great insight in this area in the future11. This collection of PPI methods will help researchers tackle this important challenge while avoiding potential pitfalls.

jove methods collection highlights: protein-protein interactions

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JoVE Methods Collection Highlights: Protein-Protein Interactions

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9.5K vistas.  University of Westminster. JoVE Methods Collection Highlights: Protein-Protein Interactions

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December 2011: This Month in JoVE

The Journal of Visualized Experiments (JoVE) closes 2011 with a December issue that begins by honoring World's AIDS Day with an article that deals with visualizing the structure of HIV envelope glycoproteins, which are a critical component of the viral infection pathway.
One key method for determining virion envelope structure is cryo-electron tomography. Prior to using this technique, soluble virus must be vitrified on a gold grid - a process that involves adding gold to a viral suspension, transferring that suspension to the grid, and plunging the grid into liquid ethane. The virus particles, present in a thin film of liquid atop the grid, are frozen at a rate greater than 100,000 K per second. Following vitrification, collected samples are loaded into the electron microscope and precisely defined regions on the grid are hit with a focused electron beam, while the grid is tilted. The tilt series that results from these imaging sessions is comprised of hundreds of individual images, which must be aligned and averaged to resolve individual envelope proteins. This computationally intense process often requires computer clusters, capable of remote-access parallel processing, like NIH's Biowulf.
Once completed, 3D models can not only provide insight into the structure of envelope glycoproteins themselves, but can also reveal the manner in which they interact other proteins, like the neutralizing antibody B12, which is information that can greatly influence vaccine design.
This article, from the laboratory of Dr. Sriram Subramaniam involves contributions from twenty authors, including not only graduates students and postdocs, but also middle and highschool students, showing that you are never too young to make an impact in the fight against a disease that has infected over 60 million people, worldwide.
In Clinical and Translational Medicine, JoVE presents an article from the University College London and the University of Oxford that investigates the physiological correlates of nociception, or pain sensation, in infants. Typically, infants respond to noxious stimuli by crying and changing their facial expressions - reactions considered to be autonomic and reliant on subcortical regions of the brain.
In order to understand the involvement of the cerebral cortex and spinal cord in infant nociception our authors first prepare infants for EEG and EMG recordings. They also set up physiological monitoring and video recording, so that the infant's facial expressions can be linked to physiological data. Following assessment of infant responses to neutral tactile stimuli, the baby is subjected to heel lancing - a clinical procedure used to obtain blood samples from infants. After data processing, electrical potentials can be compared between noxious and non-noxious stimuli in these infants, which will hopefully lead to meaningful conclusions, regarding how nociceptive information is interpreted by newborns.
JoVE's Bioengineering section takes a more clinical focus in December with an article from the University of Wisconsin, Madison, which involves the fabrication of a compartmentalized microfluidic device for studying cancer stem cell migration. Following device design, master molds are created by photolithography for the application of poly dimethyl siloxane (PDMS)- an organosilicon compound that is transparent and biologically inert - making it suitable for microfluidics applications with live cells. Specifically, our authors load tumor stem cells derived from multiform glioblastoma, a highly malignant brain cancer, into the compartmentalized devices and then place them in a specialized imaging system that acquires data over a few days. 
Time lapse video from these authors show that cancer stem cells are able to regulate their morphology in the size-constrained microfluidic channels much like they would in the interstitial space, and fluidic isolation made possible by chambers like these facilitates drug screening for potential therapeutics that can affect cancer stem cell migration.
Late on in the month, JoVE travels to France, where investigators from the Institute Pasteur and the Centre National de la Recherche Scientifique, illustrate a method for inducing expression of channel-rhodopsin, a light-activated ion channel, into the olfactory bulb of mice, as well as subsequent neuronal stimulation using a miniature light emitting diode (LED).
Our authors give detailed step-by-step instructions for preparing injection needles, and for precise injection of virus into the rostral migratory stream, a site of migration for adult born neuroblasts. Furthermore, they show how miniature LED stimulation devices are fabricated, calibrated, and implanted atop a cranial window prepared over the bulb. In particular, our authors are interested in the way that adult born neurons integrate into existing olfactory neuronal circuits, and they show that LED stimulation in awake behaving animals, can lead to activation of virally-tranduced neuroblasts in the olfactory bulb, which have migrated from the rostral migratory stream.
These video-articles comprise JoVE's highlights for the month of December. Other noteworthy upcoming articles illustrate methods for patterning conductive inks into microelectrodes, measuring cytosolic calcium in contractile lymphatics, and imaging neuronal responses to pheromones in the vomeronasal organ.

Here are some highlights from the December 2011 Issue of Journal of Visualized Experiments (JoVE).

Este mes de diciembre en JoVe

The Journal of Visualized Experiments (JoVE) closes 2011 with a December issue that begins by honoring World's AIDS Day with an article that deals with ...

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October 2012: This Month in JoVE

Here are some highlights from the October 2012 issue of Journal of Visualized Experiments (JoVE). Gr&oslash;nlund et al. demonstrate how to isolate and analyze specific cell types in plant leaves expressing green fluorescent protein (GFP) using fluorescence-activated cell sorting (FACS). This method overcomes the interfering fluorescence of the chlorophyll found in leaves, and distinguishes GFP-expressing protoplasts from non-GFP protoplasts. In JoVE Neuroscience, Babona-Pilipos et al. demonstrate how to construct a chamber for measuring galvanotaxis, or cell migration within an electric field. Through time-lapse imaging and image analysis, the authors can study the migratory behavior of neural precursor cells in an electric field, which may lead to the use of electrical stimulation to direct neural precursors to sites of injury or disease. Articles involving microfluidic platforms have a significant publication history in JoVE. Harris et al. have released three articles, which involve a microfluidic device for separating axons from neuronal cell bodies. This month in JoVE Neuroscience, Higashimori et al. use this microfluidic platform to investigate the interactions between neuronal axons and glial cells, which are critical for the physiological function of the nervous system. In JoVE Bioengineering, two research groups demonstrate the novel bioadhesive properties of chitosan-a polymer derived from chitin, which is found in fungal cell walls or in the exoskeletons of crustaceans and insects. Chitosan is used in many industrial and agricultural applications, and bioengineers are also finding uses for it in surgical applications. Foster et al. have developed a laser-activated surgical film called Surgilux, which combines chitosan with indocyanine green ICG), a photosensitive dye. This surgical film binds strongly to tissue, such as muscle, after laser irradiation. Lauto et al. developed a surgical film that combines chitosan with the photoactive dye, rose Bengal. This novel film also binds strongly to tissues, such as intestine, after it is irradiated. These adhesive films are biocompatible and may potentially be used in various surgical procedures in place of sutures. In JoVE Clinical and Translational Medicine, Fiema et al. demonstrate a high-throughput technique for validating biomarkers in graft vs. host disease, a common and life-threatening complication of cell or tissue transplantation. The authors use commercially available ELISAs to analyze multiple proteins in sequential fashion. In JoVE Immunology and Infection, Keyel et al. demonstrate how to measure the real-time kinetics of immune cell responses to bacterial toxins using high-speed live cell microscopy. This method can be used to show how immune cells respond to bacterial toxins. Combined with high-speed 3D confocal microscopy, this technique can also visualize the cellular repair response. In JoVE Applied Physics, Borisenko et al. determine the electronic structure of complex materials using angle-resolved photoemission spectroscopy in a synchotron radiation facility. By combining recent advances in synchotron radiation, surface science, and cryogenics, this method can gain a precise picture of the energy and momentum of electrons inside a solid, and address key questions in the field of condensed matter physics. This preview summarizes just a few of the notable video-articles available in the October 2012 issue of JoVE. For additional videos, please visit <a href="http://www.jove.com">www.jove.com</a>.

Here are some highlights from the October 2012 Issue of Journal of Visualized Experiments (JoVE).

Octubre 2012: Este mes en la JoVE

Here are some highlights from the October 2012 issue of Journal of Visualized Experiments (JoVE).  Gr&oslash;nlund et al. demonstrate how to isolate and ...

2012: A Year In Review

Here's a look at some of the milestones and highlights of the year 2012 in Journal of Visualized Experiments (JoVE).
At the start of 2012, JoVE reached a major milestone by publishing our 1500th article. In this publication, in JoVE Bioengineering, Yu et al. described a method for analyzing the free radical composition of cigarette smoke. This method simulated cigarette puffing with a single-port smoking device, and combined it with electron spin resonance (ESR) spectroscopy to measure the degree to which antioxidant compounds can scavenge free radicals in cigarette smoke. A possible outcome of this research is the creation of less harmful cigarettes.
JoVE Bioengineering also contained the most-viewed article of 2012. Owczarczak et al. described a method for hacking a standard inkjet printer, loading ink cartridges with a cell-suspension "bioink," and printing lines of live cells onto glass slides. The thermal inkjet printing process creates temporary pores in the cell membrane, visualized by the incorporation of fluorescently labeled actin monomers. This concept, called "bioprinting," has many potential applications in cell and tissue engineering.
Also in 2012, JoVE Bioengineering featured a protocol by Hsia et al. for isolating and purifying spider silk protein, spinning it into fibers, and assessing the fiber strength. This protocol for laboratory-scale production of spider silk, which is stronger than tensile steel, can potentially be extended to large-scale manufacturing.
In JoVE Clinical and Translational Medicine, we featured a number of articles related to stroke, one of which. In one article, M&ouml;bius-Winkler et al. demonstrated placement of the WATCHMAN left atrial appendage occlusion device from Boston Scientific. This device is designed to prevent strokes by trapping clots within the left atrial appendage of the heart before they exit. Our authors demonstrated how to insert the guide wire through the femoral artery into the heart, advance the device into the left atrium, and deploy the device in the left atrial appendage.
Another article focused on stroke, Hamel et al. described the use of a driving simulator to study how patients compensate for visual field defects following stroke. By analyzing the compensatory gaze behavior of patients as they navigated through virtual driving courses with varying degrees of complexity, our authors see great potential for the use of driving simulators in stroke rehabilitation.
In JoVE Immunology and Infection, we published an article by Keyel et al. describing the real-time kinetics of immune cell responses to bacterial toxins using live cell microscopy. Combined with high-speed 3D confocal microscopy, this technique can also visualize the cellular repair response.
Also in JoVE Immunology and Infection, we featured an article by Yap et al. describing methods for diagnosing helminth infections in children. Helminths are parasitic worms that can infect the intestinal tract of humans and animals, and may be distinguished based on their morphology. The same article demonstrated how to measure the impact of helminth infections on physical fitness in children, which is a measure of overall health.
In JoVE Neuroscience, we featured a method by Hoffmann et al., who used tiny headphones to alter the auditory feedback in songbirds, and analyzed the computational and neurophysiological basis of vocal learning in birds when they adjust their singing in response to altered acoustic signals.
Also in JoVE Neuroscience, we moved from birds to bees with a method that assesses associative and non-associative tactile learning in honeybees. This method, by Mujagi&#263; et al., allowed bees to scan different metal surfaces with their antennae, then conditioned them to expect sugar water when their antennae touched particular surfaces, and analyzed the corresponding changes in antennal movement.
In 2012, we launched a brand-new section: JoVE Applied Physics, which features articles on subjects ranging from plasma physics to material science. The Applied Physics section also contained JoVE's 2000th video article, filmed in a synchrotron radiation facility, where Borisenko et al. showed how to determine the electronic structure of complex materials using angle-resolved photoemission spectroscopy.
In the JoVE General section, we published an article by Artioli et al. demonstrating the use of a portable gas analyzer to measure oxygen consumption during complex exercise - such as judo. By analyzing oxygen consumption and collecting blood for measuring plasma lactate concentration, this method can determine the relative contributions of different energy systems to specific aspects of complex exercise.
Finally, JoVE followed Dolhi et al. to a permanently ice-covered saline lake in Antarctica. There, scientists drilled through the ice to study single-celled microorganisms called protists that live in the harsh conditions of Antarctica.
This Year in Review was just a brief glimpse of a few of over 600 video articles that JoVE offered in 2012. Browse the JoVE archives to see thousands of other videos, and stay tuned for what's coming up this year in JoVE: The Journal of Visualized Experiments.

Here's a look at some of the milestones and highlights of the year 2012 in Journal of Visualized Experiments (JoVE).

2012: Un año en revisión

 
 
Here's a look at some of the milestones and highlights of the year 2012 in Journal of Visualized Experiments (JoVE).
At the start of 2012, JoVE ...