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Current Protocols in Chemical Biology

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https://www.readbyqxmd.com/read/28628203/photoactivated-in-vivo-proximity-labeling
#1
David B Beck, Roberto Bonasio
Identification of molecular interactions is paramount to understanding how cells function. Most available technologies rely on co-purification of a protein of interest and its binding partners. Therefore, they are limited in their ability to detect low-affinity interactions and cannot be applied to proteins that localize to difficult-to-solubilize cellular compartments. In vivo proximity labeling (IPL) overcomes these obstacles by covalently tagging proteins and RNAs based on their proximity in vivo to a protein of interest...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28628202/paper-based-invasion-assays-for-quantifying-cellular-movement-in-three-dimensional-tissue-like-structures
#2
C Chad Lloyd, Matthew W Boyce, Matthew R Lockett
To elucidate the chemical and environmental conditions that promote invasion of cancer cells, an assay is needed in which the chemical landscape of a tumor-like environment can be experimentally manipulated and probed. The three-dimensional paper-based invasion assays described here simulate poorly vascularized tissue and allow the invasion of cancerous cells to be visualized and quantified. These cultures are easy to assemble and allow multiple invasion assays to be performed in parallel. By using different materials to control gradients formed across the culture, the chemotactic potential of small molecules can be evaluated in a more representative tissue microenvironment...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28628201/designing-drug-response-experiments-and-quantifying-their-results
#3
Marc Hafner, Mario Niepel, Kartik Subramanian, Peter K Sorger
We developed a Python package to help in performing drug-response experiments at medium and high throughput and evaluating sensitivity metrics from the resulting data. In this article, we describe the steps involved in (1) generating files necessary for treating cells with the HP D300 drug dispenser, by pin transfer or by manual pipetting; (2) merging the data generated by high-throughput slide scanners, such as the Perkin Elmer Operetta, with treatment annotations; and (3) analyzing the results to obtain data normalized to untreated controls and sensitivity metrics such as IC50 or GR50 ...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28628200/local-generation-and-imaging-of-hydrogen-peroxide-in-living-cells
#4
Yulia A Bogdanova, Carsten Schultz, Vsevolod V Belousov
Described here is a localized H2 O2 generation-detection system consisting of a yeast D-amino acid oxidase (DAAO) and two spectrally distinct variants of biosensor, HyPer2 and HyPerRed based on circularly permutated yellow and red fluorescent proteins, respectively, which enables spatiotemporal production and examination of the intracellular H2 O2 dynamics. The protocol describes using this system in a simple cell culture model. We provide detailed instructions on imaging of H2 O2 generated by the activated DAAO...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28628199/measuring-cancer-drug-sensitivity-and-resistance-in-cultured-cells
#5
Mario Niepel, Marc Hafner, Mirra Chung, Peter K Sorger
Measuring the potencies of small-molecule drugs in cell lines is a critical aspect of preclinical pharmacology. Such experiments are also prototypical of high-throughput experiments in multi-well plates. The procedure is simple in principle, but many unrecognized factors can affect the results, potentially making data unreliable. The procedures for measuring drug response described here were developed by the NIH LINCS program to improve reproducibility. Key features include maximizing uniform cell growth during the assay period, accounting for the effects of cell density on response, and correcting sensitivity measures for differences in proliferation rates...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28253435/detecting-membrane-protein-protein-interactions-using-the-mammalian-membrane-two-hybrid-mamth-assay
#6
Punit Saraon, Ingrid Grozavu, Sang Hyun Lim, Jamie Snider, Zhong Yao, Igor Stagljar
Protein-protein interactions (PPIs) play an integral role in numerous cellular processes. Membrane protein interactions, in particular, are critical in cellular responses to stresses and stimuli, with dysfunction of these PPIs (e.g., due to aberrant expression and/or mutation of interaction partners) leading to a diverse array of pathological states. Exploration of the interaction space and dynamics of membrane proteins is difficult due to the limitations of current techniques used to study proteins in the biochemically complex environment of biological membranes...
March 2, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28253434/a-general-non-radioactive-atpase-assay-for-chromatin-remodeling-complexes
#7
Benjamin Z Stanton, Courtney Hodges, Gerald R Crabtree, Keji Zhao
Chromatin remodeling complexes couple the energy released from ATP hydrolysis to facilitate transcription, recombination, and repair mechanisms essential for a wide variety of biologic responses. While recombinant expression of the regulatory subunits of these enzymes is possible, measuring catalytic (ATPase) activity of the intact complexes recovered from normal or mutant cells is critical for understanding their mechanisms. SWI/SNF-like remodeling complexes can be megadaltons in size and include many regulatory subunits, making reconstitution of purified subunits challenging for recapitulating in vivo function...
March 2, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28253433/ubfluor-a-fluorescent-thioester-to-monitor-hect-e3-ligase-catalysis
#8
David T Krist, Peter K Foote, Alexander V Statsyuk
HECT E3 ubiquitin ligases (∼28 are known) are associated with many phenotypes in eukaryotes and are important drug targets. However, assays used to screen for small molecule inhibitors of HECT E3s are complex and require ATP, Ub, E1, E2, and HECT E3 enzymes, producing three covalent thioester enzyme intermediates E1∼Ub, E2∼Ub, and HECT E3∼Ub (where ∼ indicates a thioester bond), and mixtures of polyubiquitin chains. To reduce the complexity of the assay, we developed a novel class of fluorescent probes, UbFluor, that act as mechanistically relevant pseudosubstrates of HECT E3s...
March 2, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27925669/biochemical-biophysical-and-cellular-techniques-to-study-the-guanine-nucleotide-exchange-factor-giv-girdin
#9
Pradipta Ghosh, Nicolas Aznar, Lee Swanson, I-Chung Lo, Inmaculada Lopez-Sanchez, Jason Ear, Cristina Rohena, Nicholas Kalogriopoulos, Linda Joosen, Ying Dunkel, Nina Sun, Peter Nguyen, Deepali Bhandari
Canonical signal transduction via heterotrimeric G proteins is spatiotemporally restricted, i.e., triggered exclusively at the plasma membrane, only by agonist activation of G protein-coupled receptors via a finite process that is terminated within a few hundred milliseconds. Recently, a rapidly emerging paradigm has revealed a noncanonical pathway for activation of heterotrimeric G proteins via the nonreceptor guanidine-nucleotide exchange factor, GIV/Girdin. Biochemical, biophysical, and functional studies evaluating this pathway have unraveled its unique properties and distinctive spatiotemporal features...
December 7, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27925668/cyclic-immunofluorescence-cycif-a-highly-multiplexed-method-for-single-cell-imaging
#10
Jia-Ren Lin, Mohammad Fallahi-Sichani, Jia-Yun Chen, Peter K Sorger
Cyclic Immunofluorescence (CycIF) is a public-domain method for performing highly multiplexed immunofluorescence imaging using a conventional epifluorescence microscope. It uses simple reagents and existing antibodies to construct images with up to 30 channels by sequential 4- to 6-channel imaging followed by fluorophore inactivation. Three variant methods are described, the most generally useful of which involves staining fixed cells with antibodies directly conjugated to Alexa Fluor dyes and imaging in four colors, inactivating fluorophores using a mild base in the presence of hydrogen peroxide and light, and then performing another round of staining and imaging...
December 7, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27925667/tracking-the-activity-of-mtorc1-in-living-cells-using-genetically-encoded-fret-based-biosensor-torcar
#11
Xin Zhou, Simin Li, Jin Zhang
Mechanistic target of rapamycin complex 1 (mTORC1) is a highly conserved serine/threonine protein kinase that responds to multiple distinct signals (e.g., growth factors, amino acids, stress, and energy level) and coordinates cell growth and proliferation. The underlying molecular mechanisms by which these stimuli regulate the activity of mTORC1 are still not fully understood. The spatial compartmentalization of mTORC1 signaling has been suggested as an important mechanism for mTORC1 to achieve the signal specificity and efficiency...
December 7, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27925666/enriching-s-4-u-rna-using-methane-thiosulfonate-mts-chemistry
#12
Erin E Duffy, Matthew D Simon
Metabolic labeling of cellular RNA is a useful approach to study RNA biology. 4-Thiouridine (s(4) U) is a convenient nucleoside for metabolic labeling because it is cell permeable and is incorporated into newly transcribed RNA, and the sulfur moiety provides a handle for biochemical purification. However, a critical step in the purification of s(4) U-RNA is the efficiency of the chemistry used to enrich s(4) U-RNA. Here, we present a protocol for s(4) U-RNA enrichment that includes efficient and reversible covalent chemistry to biotinylate s(4) U-RNA using the activated disulfide methane thiosulfonate conjugated to biotin (MTS-biotin), followed by enrichment on streptavidin beads...
December 7, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27622569/nanoparticle-templated-molecular-recognition-platforms-for-detection-of-biological-analytes
#13
Abraham G Beyene, Gozde S Demirer, Markita P Landry
Molecular recognition of biological analytes with optical nanosensors provides both spatial and temporal biochemical information. A recently developed sensing platform exploits near-infrared fluorescent single-wall carbon nanotubes combined with electrostatically pinned heteropolymers to yield a synthetic molecular recognition technique that is maximally transparent through biological matter. This molecular recognition technique is known as corona phase molecular recognition (CoPhMoRe). In CoPhMoRe, the specificity of a folded polymer toward an analyte does not arise from a pre-existing polymer-analyte chemical affinity...
September 13, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27622568/reverse-phase-protein-arrays-for-compound-profiling
#14
Nathan Moerke, Mohammad Fallahi-Sichani
Reverse phase protein arrays (RPPAs), also called reverse phase lysate arrays (RPLAs), involve immobilizing cell or tissue lysates, in small spots, onto solid supports which are then probed with primary antibodies specific for proteins or post-translational modifications of interest. RPPA assays are well suited for large-scale, high-throughput measurement of protein and PTM levels in cells and tissues. RPPAs are affordable and highly multiplexable, as a large number of arrays can readily be produced in parallel and then probed separately with distinct primary antibodies...
September 13, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27622567/fabrication-of-3-d-reconstituted-organoid-arrays-by-dna-programmed-assembly-of-cells-dpac
#15
Michael E Todhunter, Robert J Weber, Justin Farlow, Noel Y Jee, Alec E Cerchiari, Zev J Gartner
Tissues are the organizational units of function in metazoan organisms. Tissues comprise an assortment of cellular building blocks, soluble factors, and extracellular matrix (ECM) composed into specific three-dimensional (3-D) structures. The capacity to reconstitute tissues in vitro with the structural complexity observed in vivo is key to understanding processes such as morphogenesis, homeostasis, and disease. In this article, we describe DNA-programmed assembly of cells (DPAC), a method to fabricate viable, functional arrays of organoid-like tissues within 3-D ECM gels...
September 13, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27258691/optogenetic-control-of-nuclear-protein-import-in-living-cells-using-light-inducible-nuclear-localization-signals-linus
#16
Pierre Wehler, Dominik Niopek, Roland Eils, Barbara Di Ventura
Many biological processes are regulated by the timely import of specific proteins into the nucleus. The ability to spatiotemporally control the nuclear import of proteins of interest therefore allows study of their role in a given biological process as well as controlling this process in space and time. The light-inducible nuclear localization signal (LINuS) was developed based on a natural plant photoreceptor that reversibly triggers the import of proteins of interest into the nucleus with blue light. Each LINuS is a small, genetically encoded domain that is fused to the protein of interest at the N or C terminus...
June 2, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27258690/combinatorial-library-screening-coupled-to-mass-spectrometry-to-identify-valuable-cyclic-peptides
#17
Silvia A Camperi, Silvana L Giudicessi, María C Martínez-Ceron, Juan M Gurevich-Messina, Soledad L Saavedra, Gerardo Acosta, Osvaldo Cascone, Rosa Erra-Balsells, Fernando Albericio
Combinatorial library screening coupled to mass spectrometry (MS) analysis is a practical approach to identify useful peptides. Cyclic peptides can have high biological activity, selectivity, and affinity for target proteins, and high stability against proteolytic degradation. Here we describe two strategies to prepare combinatorial libraries suitable for MS analysis to accelerate the discovery of cyclic peptide structures. Both approaches use ChemMatrix resin and the linker 4-hydroxymethylbenzoic acid. One strategy involves the synthesis of a one-bead-two-peptides library in which each bead contains both the cyclic peptide and its linear counterpart to facilitate MS analysis...
June 2, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27258689/liquid-exfoliation-of-layered-transition-metal-dichalcogenides-for-biological-applications
#18
Emily P Nguyen, Torben Daeneke, Serge Zhuiykov, Kourosh Kalantar-Zadeh
Known to possess distinctive properties that differ greatly from their bulk form, layered two-dimensional materials have been extensively studied and incorporated into many versatile applications ranging from optoelectronics to sensors. For biomedical research, two-dimensional transition metal dichalcogenides (2D TMDs) have garnered much interest as they have been shown to exhibit relatively low toxicity, high stability in aqueous environments, and the ability to adhere to biological materials such as proteins...
June 2, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27258688/pseudo-ligandless-click-chemistry-for-oligonucleotide-conjugation
#19
Stephanie Mack, Munira F Fouz, Sourav Kumar Dey, Subha R Das
Particularly for its use in bioconjugations, the copper-catalyzed (or copper-promoted) azide-alkyne cycloaddition (CuAAC) reaction or 'click chemistry', has become an essential component of the modern chemical biologist's toolbox. Click chemistry has been applied to DNA, and more recently, RNA conjugations, and the protocols presented here can be used for either. The reaction can be carried out in aqueous buffer, and uses acetonitrile as a minor co-solvent that serves as a ligand to stabilize the copper. The method also includes details on the analysis of the reaction product...
June 2, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/26995354/isotope-targeted-glycoproteomics-isotag-to-characterize-intact-metabolically-labeled-glycopeptides-from-complex-proteomes
#20
Christina M Woo, Carolyn R Bertozzi
Protein glycosylation plays many critical roles in biological function and creates the most diversity of all post-translational modifications (PTMs). Glycan structural diversity is directly correlated with difficulty in characterizing the intact glycoproteome by mass spectrometry (MS). In this protocol, we describe a novel mass-independent chemical glycoproteomics platform for characterizing intact, metabolically labeled glycopeptides from complex proteomes, termed Isotope Targeted Glycoproteomics (IsoTaG)...
March 16, 2016: Current Protocols in Chemical Biology
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