Methods Collections

Protein-Protein Interaction Methods

Collection Overview

The study of protein-protein interactions is key to understanding the network of molecular interactions underlying biological effects, and defining how individual proteins perform their functional role within a cell.  Detailed understanding of these interactions often provides  insight into aberrant signaling in disease.  This collection brings together the wide range of techniques that have been developed to study interactions using in vitro, cell-based and in vivo approaches. Ea Show More



Calibration-Free in-vitro Quantification of Homo-Oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Rory Nolan1, Luis A. Alvarez1, Samuel C. Griffiths2, Jonathan Elegheert2, Christian Siebold2, Sergi Padilla-Parra*1, 2, 3, 4
1Cellular Imaging Group, Wellcome Centre Human Genetics, , University of Oxford, Oxford, 2Division of Structural Biology, Wellcome Centre Human Genetics, University of Oxford, Oxford, 3Dynamic Structural Virology Group, Biocruces Health Research Centre, Barakaldo, Spain, 4IKERBASQUE, Basque Foundation for Science, Bilbao, Spain

We present a calibration free approach to detect protein homo-dimerization and oligomerization that employs fluorescence fluctuation spectroscopy (FFS) using a conventional confocal microscope equipped with digital detectors. Using a purified mVenus-labelled FKBP12 before and after the application of a dimerization drug (AP20187, referred commercially as BB, Clonetech); we show that number and brightness accurately provide information on the oligomeric state of FKBP12 when using very low concent Show More

Structural Studies of Macromolecular Interactions in Solution

Michael Overduin*1, Steffane McLelland1, Trushar R Patel2
1University of Alberta, Edmonton, Canada, 2University of Lethbridge Faculty of Arts and Science, Alberta, Canada

Proteins containing multiple structural domains that interact with their binding partners (e.g. proteins and nucleic acids) present technical challenges for determining how such macromolecular assemblies are formed. An emerging tool that can be used to elucidate which specific domains mediate interactions in multicomponent systems is described. A method for calculating hybrid solution structures of such assemblies is provided, and integrates data from small angle X-ray scattering (SAXS Show More

Co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions.

Xiaofeng Zheng*1, Calvin Qing Wei Ho2, Xiaowei Zheng3, Kian Leong Lee4, Katarina Gradin5, Teresa S. Pereira3, Yusuf Ali6
1 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, 2Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, 3The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden, 4 Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore, 5Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden, 6Singapore Eye Research Institute (SERI), Singapore General Hospital, Singapore

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated α subunit (HIF-1α) and constitutively expressed β subunit (HIF-1β) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. 

The identification Show More

Measuring interactions of globular and filamentous proteins by nuclear magnetic resonance spectroscopy and microscale thermophoresis

Michael Overduin*1
1University of Alberta, Edmonton, Canada

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. This protocol describes a method for detecting and measuring such interactions using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The glob Show More

Detection of Heterodimerization of Protein Isoforms using an in situ Proximity Ligation Assay.

Sofiia Karchugina1, Jonathan Chernoff*1
1Fox Chase Cancer Center, Philadelphia, PA, United States

Dysregulation or loss of MST1/Hippo signaling is linked to developmental disorders and carcinogenesis with poor prognosis1. In mammals, the kinases MST1 and MST2 activate (phosphorylate) MOB1 and LATS1/2, which then phosphorylates and inactivates the transcriptional co-activator Yes-associated protein (YAP)2. In its active (unphosphorylated) form, YAP acts as an oncogene and enhances transcription of genes involved in cell proliferation, and inactivation of YAP by the Hippo pathway suppresses ce Show More

2 in 1: One-step affinity purification for the parallel analysis of protein–protein and protein–metabolite complexes

Marcin Luzarowski1, Izabela Wojciechowska1, Aleksandra Skirycz*1
1Max Planck Institute of Molecular Plant Physiology, Golm, Germany

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein–protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein–metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our pr Show More

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