scholarly journals Lanthanide-Based Imaging of Protein–Protein Interactions in Live Cells

2013 ◽  
Vol 53 (4) ◽  
pp. 1839-1853 ◽  
Author(s):  
Megha Rajendran ◽  
Engin Yapici ◽  
Lawrence W. Miller
Keyword(s):  
Protein Interactions ◽  
Live Cells ◽  
Protein Protein Interactions

scholarly journals Assay Systems for Profiling Deubiquitinating Activity

2020 ◽  
Vol 21 (16) ◽  
pp. 5638
Author(s):  
Jinhong Cho ◽  
Jinyoung Park ◽  
Eunice EunKyeong Kim ◽  
Eun Joo Song
Keyword(s):  
Protein Degradation ◽  
Protein Interactions ◽  
Live Cells ◽  
Deubiquitinating Enzyme ◽  
Protein Protein Interactions ◽  
Deubiquitinating Enzymes ◽  
Cell Lysates ◽  
Cellular Processes

Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein–protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo.

scholarly journals Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1070 ◽  
Author(s):  
Danielle G. May ◽  
Kelsey L. Scott ◽  
Alexandre R. Campos ◽  
Kyle J. Roux
Keyword(s):  
Protein Interactions ◽  
Culture Media ◽  
Affinity Purification ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Experimental Protocol ◽  
Established Method ◽  
Cellular Toxicity ◽  
Practical Applications ◽  
Protein Instability

BioID is a well-established method for identifying protein–protein interactions and has been utilized within live cells and several animal models. However, the conventional labeling period requires 15–18 h for robust biotinylation which may not be ideal for some applications. Recently, two new ligases termed TurboID and miniTurbo were developed using directed evolution of the BioID ligase and were able to produce robust biotinylation following a 10 min incubation with excess biotin. However, there is reported concern about inducibility of biotinylation, cellular toxicity, and ligase stability. To further investigate the practical applications of TurboID and ascertain strengths and weaknesses compared to BioID, we developed several stable cell lines expressing BioID and TurboID fusion proteins and analyzed them via immunoblot, immunofluorescence, and biotin-affinity purification-based proteomics. For TurboID we observed signs of protein instability, persistent biotinylation in the absence of exogenous biotin, and an increase in the practical labeling radius. However, TurboID enabled robust biotinylation in the endoplasmic reticulum lumen compared to BioID. Induction of biotinylation could be achieved by combining doxycycline-inducible expression with growth in biotin depleted culture media. These studies should help inform investigators utilizing BioID-based methods as to the appropriate ligase and experimental protocol for their particular needs.

scholarly journals Enabling systematic interrogation of protein–protein interactions in live cells with a versatile ultra-high-throughput biosensor platform

2015 ◽  
Vol 8 (3) ◽  
pp. 271-281 ◽  
Author(s):  
Xiu-Lei Mo ◽  
Yin Luo ◽  
Andrei A. Ivanov ◽  
Rina Su ◽  
Jonathan J. Havel ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
Live Cells ◽  
Protein Protein Interactions

QUANTITATIVE FRET MEASUREMENT BASED ON CONFOCAL MICROSCOPY IMAGING AND PARTIAL ACCEPTOR PHOTOBLEACHING

2012 ◽  
Vol 05 (03) ◽  
pp. 1250015 ◽  
Author(s):  
XIAO-PING WANG ◽  
HUAI-NA YU ◽  
TONG-SHENG CHEN
Keyword(s):  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Region Of Interest ◽  
Living Cells ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Microscopy Imaging ◽  
Acceptor Photobleaching ◽  
Induced Apoptosis

Fluorescence resonance energy transfer (FRET) technology had been widely used to study protein–protein interactions in living cells. In this study, we developed a ROI-PbFRET method to real-time quantitate the FRET efficiency of FRET construct in living cells by combining the region of interest (ROI) function of confocal microscope and partial acceptor photobleaching. We validated the ROI-PbFRET method using GFPs-based FRET constructs including 18AA and SCAT3, and used it to quantitatively monitor the dynamics of caspase-3 activation in single live cells stably expressing SCAT3 during staurosporine (STS)-induced apoptosis. Our results for the first demonstrate that ROI-PbFRET method is a powerful potential tool for detecting the dynamics of molecular interactions in live cells.

scholarly journals Optogenetic control of mRNA localization and translation in live cells

2020 ◽  
Author(s):  
Sangkyu Lee ◽  
Won Heo ◽  
Na Kim
Keyword(s):  
Protein Synthesis ◽  
Blue Light ◽  
Protein Interactions ◽  
Mrna Localization ◽  
Spatiotemporal Dynamics ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Protein Clusters ◽  
Specific Mrnas ◽  
Specific Mrna

Abstract Numerous efforts have been made toward the goal of visualizing the spatiotemporal dynamics of single mRNA molecules, yet our capacity for precisely controlling their functions lags behind. Here, we present an optogenetic approach for manipulating the localization and translation of specific mRNAs in live cells. Our technique combines blue light-responsive protein-protein interactions with mRNA visualization modules to robustly and reversibly generate protein clusters that can trap specific mRNA molecules. This sequestration reduces the binding chance of mRNAs with ribosomes, thereby dramatically attenuating protein synthesis

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