| 1. |
- Aili, Margareta, et al.
(författare)
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GAP activity of Yersinia YopE
- 2002
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Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 358, s. 359-70
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Tidskriftsartikel (refereegranskat)
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| 2. |
- Aspholm, Marina, et al.
(författare)
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Helicobacter pylori adhesion to carbohydrates
- 2006
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Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 417, s. 293-339
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Tidskriftsartikel (refereegranskat)abstract
- Adherence of bacterial pathogens to host tissues contributes to colonization and virulence and typically involves specific interactions between bacterial proteins called adhesins and cognate oligosaccharide (glycan) or protein motifs in the host that are used as receptors. A given pathogen may have multiple adhesins, each specific for a different set of receptors and, potentially, with different roles in infection and disease. This chapter provides strategies for identifying and analyzing host glycan receptors and the bacterial adhesins that exploit them as receptors, with particular reference to adherence of the gastric pathogen Helicobacter pylori.
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| 3. |
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| 4. |
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| 5. |
- Borga, Magnus, et al.
(författare)
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Brown adipose tissue in humans: detection and functional analysis using PET (positron emission tomography), MRI (magnetic resonance imaging), and DECT (dual energy computed tomography).
- 2014
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Ingår i: Methods in enzymology. - : Elsevier. - 1557-7988. ; 537, s. 141-59, s. 141-159
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Tidskriftsartikel (refereegranskat)abstract
- If the beneficial effects of brown adipose tissue (BAT) on whole body metabolism, as observed in nonhuman experimental models, are to be translated to humans, tools that accurately measure how BAT influences human metabolism will be required. This chapter discusses such techniques, how they can be used, what they can measure and also some of their limitations. The focus is on detection and functional analysis of human BAT and how this can be facilitated by applying advanced imaging technology such as positron emission tomography, magnetic resonance imaging, and dual energy computed tomography.
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| 6. |
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| 7. |
- Budayova-Spano, Monika, et al.
(författare)
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Large crystal growth for neutron protein crystallography
- 2020
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Ingår i: Neutron Crystallography in Structural Biology. - : Elsevier. - 1557-7988 .- 0076-6879. ; 634, s. 21-46
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Bokkapitel (refereegranskat)abstract
- The use of neutron protein crystallography (NPX) is expanding rapidly, with most structures determined in the last decade. This growth is stimulated by a number of developments, spanning from the building of new NPX beamlines to the availability of improved software for structure refinement. The main bottleneck preventing structural biologists from adding NPX to the suite of methods commonly used is the large volume of the individual crystals required for a successful experiment. A survey of deposited NPX structures in the Protein Data Bank shows that about two-thirds came from crystals prepared using vapor diffusion, while batch and dialysis-based methods all-together contribute to most of the remaining one-third. This chapter explains the underlying principles of these protein crystallization methods and provides practical examples that may help others to successfully prepare large crystals for NPX.
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| 8. |
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| 9. |
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| 10. |
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| 11. |
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| 12. |
- Eklof, Jens M., et al.
(författare)
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Distinguishing xyloglucanase activity in endo-β(1 → 4)glucanases
- 2012
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Ingår i: Methods in Enzymology. - : Elsevier BV. - 0076-6879 .- 1557-7988. - 9780124159310 ; 510, s. 97-120
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Tidskriftsartikel (refereegranskat)abstract
- The ability of beta-glucanases to cleave xyloglucans, a family of highly decorated beta-glucans ubiquitous in plant biomass, has traditionally been overlooked in functional biochemical studies. An emerging body of data indicates, however, that a spectrum of xyloglucan specificity resides in diverse glycoside hydrolases from a range of carbohydrate-active enzyme families including classic "cellulase" families. This chapter outlines a series of enzyme kinetic and product analysis methods to establish degrees of xyloglucan specificity and modes of action of glycosidases emerging from enzyme discovery projects.
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| 13. |
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| 14. |
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| 15. |
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| 16. |
- Galluzzi, L, et al.
(författare)
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Preface
- 2017
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Ingår i: Methods in enzymology. - 1557-7988. ; 588, s. XXV-XXXI
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 17. |
- Galluzzi, L, et al.
(författare)
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Preface
- 2017
-
Ingår i: Methods in enzymology. - 1557-7988. ; 587, s. XXIII-XXIX
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 18. |
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| 19. |
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| 20. |
- Gyllensten, Ulf, et al.
(författare)
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Sequencing of in vitro amplified DNA
- 1993
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Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 218, s. 3-16
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 21. |
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| 22. |
- He, Feng, et al.
(författare)
-
Chapter 6. Qualitative and quantitative assessment of the activity of the yeast nonsense-mediated mRNA decay pathway.
- 2008
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Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 449, s. 127-47
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Saccharomyces cerevisiae provides an ideal model system for elucidation of the molecular mechanisms that regulate the nonsense-mediated mRNA decay (NMD) pathway. This chapter describes an array of molecular biological, genetic, and biochemical tools that facilitate the characterization of transcripts that comprise NMD substrates and provide insights into the roles of the upf/nmd proteins in mRNA decay and translation termination. Examples illustrate the use of these methods in wild-type and NMD-deficient cells to monitor the abundance, structure, and half-lives of nonsense-containing mRNAs, the read through of premature termination codons by the ribosome, and the positioning of ribosomes at or near normal and premature termination codons.
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| 23. |
- Hebert, Hans, et al.
(författare)
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Two-dimensional crystallization and electron crystallography of MAPEG proteins.
- 2005
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Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 401, s. 161-8
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Tidskriftsartikel (refereegranskat)abstract
- Members of the membrane-associated proteins in the eicosanoid and glutathione metabolism (MAPEG) superfamily have been subjected to two-dimensional crystallization experiments. A common denominator for successful attempts has been the use of a low lipid/protein ratio in the range of 1-9 (mol/mol). Electron crystallography demonstrated either hexagonal or orthorhombic packing of trimeric protein units. Three-dimensional structure analysis of the MAPEG member microsomal glutathione transferase 1 has shown that the monomer for this protein contains a left-handed bundle of four transmembrane helices. It is likely that this is a common structural motif for MAPEG proteins, because projection maps of all structurally characterized members are very similar.
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| 24. |
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| 25. |
- Heidorn, Thorsten, et al.
(författare)
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Synthetic Biology in Cyanobacteria : Engineering and Analyzing Novel Functions
- 2011
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Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 497, s. 539-579
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Forskningsöversikt (refereegranskat)abstract
- Cyanobacteria are the only prokaryotes capable of using sunlight as their energy, water as an electron donor, and air as a source of carbon and, for some nitrogen-fixing strains, nitrogen. Compared to algae and plants, cyanobacteria are much easier to genetically engineer, and many of the standard biological parts available for Synthetic Biology applications in Escherichia coli can also be used in cyanobacteria. However, characterization of such parts in cyanobacteria reveals differences in performance when compared to E. coli, emphasizing the importance of detailed characterization in the cellular context of a biological chassis. Furthermore, cyanobacteria possess special characteristics (e.g., multiple copies of their chromosomes, high content of photosynthetically active proteins in the thylakoids, the presence of exopolysaccharides and extracellular glycolipids, and the existence of a circadian rhythm) that have to be taken into account when genetically engineering them. With this chapter, the synthetic biologist is given an overview of existing biological parts, tools and protocols for the genetic engineering, and molecular analysis of cyanobacteria for Synthetic Biology applications.
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| 26. |
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| 27. |
- Hjorth-Jensen, Samuel John, et al.
(författare)
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Prospects for membrane protein crystals in NMX
- 2020
-
Ingår i: Neutron Crystallography in Structural Biology. - : Elsevier. - 1557-7988 .- 0076-6879. ; 634, s. 47-68
-
Bokkapitel (refereegranskat)abstract
- Adding hydrogen atoms and protonation states to structures of membrane proteins requires successful implementation of neutron macromolecular crystallography (NMX). This information would significantly increase our fundamental understanding of the transport processes membrane proteins undertake. To grow the large crystals needed for NMX studies requires significant amounts of stable protein, but once that challenge is overcome there is no intrinsic property of membrane proteins preventing the growth of large crystals per se. The calcium-transporting P-type ATPase (SERCA) has been thoroughly characterized biochemically and structurally over decades. We have extended our crystallization efforts to assess the feasibility of growing SERCA crystals for NMX—exploring microdialysis and capillary counterdiffusion crystallization techniques as alternatives to the traditional vapor diffusion crystallization experiment. Both methods possess crystallization dynamics favorable for maximizing crystal size and we used them to facilitate the growth of large crystals, validating these approaches for membrane protein crystallization for NMX.
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| 28. |
- Hohmann, Stefan, 1956, et al.
(författare)
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Yeast osmoregulation
- 2007
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Ingår i: Methods in Enzymology. - 1557-7988 .- 0076-6879. ; 428, s. 29-45
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Tidskriftsartikel (refereegranskat)abstract
- Osmoregulation is the active control of the cellular water balance and encompasses homeostatic mechanisms crucial for life. The osmoregulatory system in the yeast Saccharomyces cerevisiae is particularly well understood. Key to yeast osmoregulation is the production and accumulation of the compatible solute glycerol, which is partly controlled by the high osmolarity glycerol (HOG) signaling system. Genetic analyses combined with studies on protein-protein interactions have revealed the wiring scheme of the HOG signaling network, a branched mitogen-activated protein (MAP) kinase (MAPK) pathway that eventually converges on the MAPK Hog1. Hog1 is activated following cell shrinking and controls posttranscriptional processes in the cytosol as well as gene expression in the nucleus. HOG pathway activity can easily and rapidly be controlled experimentally by extracellular stimuli, and signaling and adaptation can be separated by a system of forced adaptation. This makes yeast osmoregulation suitable for studies on system properties of signaling and cellular adaptation via mathematical modeling. Computational simulations and parallel quantitative time course experimentation on different levels of the regulatory system have provided a stepping stone toward a holistic understanding, revealing how the HOG pathway can combine rigorous feedback control with maintenance of signaling competence. The abundant tools make yeast a suitable model for an integrated analysis of cellular osmoregulation. Maintenance of the cellular water balance is fundamental for life. All cells, even those in multicellular organisms with an organism-wide osmoregulation, have the ability to actively control their water balance. Osmoregulation encompasses homeostatic processes that maintain an appropriate intracellular environment for biochemical processes as well as turgor of cells and organism. In the laboratory, the osmoregulatory system is studied most conveniently as a response to osmotic shock, causing rapid and dramatic changes in the extracellular water activity. Those rapid changes mediate either water efflux (hyperosmotic shock), and hence cell shrinkage, or influx (hypoosmotic shock), causing cell swelling. The yeast S. cerevisiae, as a free-living organism experiencing both slow and rapid changes in extracellular water activity, has proven a suitable and genetically tractable experimental system in studying the underlying signaling pathways and regulatory processes governing osmoregulation. Although far from complete, the present picture of yeast osmoregulation is both extensive and detailed (de Nadal et al., 2002; Hohmann, 2002; Klipp et al., 2005). Simulations using mathematical models combined with time course measurements of different molecular processes in signaling and adaptation have allowed elucidation of the first system properties on the yeast osmoregulatory network.
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| 29. |
- Jesorka, Aldo, 1967, et al.
(författare)
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COMPLEX NANOTUBE-LIPOSOME NETWORKS
- 2009
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Ingår i: Methods in Enzymology. - 1557-7988 .- 0076-6879. ; 464:C, s. 309-325
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Tidskriftsartikel (refereegranskat)abstract
- Surfactant nanotube-vesicle networks (NVN) belong to the smallest artificial devices known to date for performing controlled chemical operations with enzymes. Newly established means for transport of chemical reactants between containers, as well as advancements in initiation and control of chemical reactions in such systems have opened pathways to new devices with a resolution down to the single-molecule level. Here, we summarize the fabrication and functionalization of complex nanotube-liposome networks for such devices, and discuss related aspects of their application for studying chemical kinetics and materials transport phenomena in ultrasmall-scale bio-mimetic environments.
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| 30. |
- Jesorka, Aldo, 1967, et al.
(författare)
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Microfluidic technology for investigation of protein function in single adherent cells
- 2019
-
Ingår i: Methods in Enzymology. - : Elsevier. - 1557-7988 .- 0076-6879. ; 628
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Instrumental techniques and associated methods for single cell analysis, designed to investigate and measure a broad range of cellular parameters in search of unique features, address key limitations of conventional cell-based assays with their ensemble average response. While many different single cell techniques exist for suspension cultures, which can process and characterize large numbers of individual cells in rapid succession, the access to surface-immobilized cells in typical 2D and 3D culture environments remains challenging. Open space microfluidics has created new possibilities in this area, allowing for exclusive access to single cells in adherent cultures, even at high confluency. In this chapter, we briefly review new microtechnologies for the investigation of protein function in single adherent cells, and present an overview over related recent applications of the multifunctional pipette (Biopen), a microfluidic multi-solution dispensing system that uses hydrodynamic confinement in open volume environments in order to establish a superfusion zone over selected single cells in adherent cultures.
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| 31. |
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| 32. |
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| 33. |
- Larsson, Per-Olof, et al.
(författare)
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Magnetically enhanced phase separation
- 1994
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Ingår i: Methods in Enzymology. - : Elsevier. - 0076-6879 .- 1557-7988. - 0121821293 ; 228, s. 112-117
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 34. |
- Lenton, Samuel, et al.
(författare)
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From dilute to concentrated solutions of intrinsically disordered proteins : Interpretation and analysis of collected data
- 2023
-
Ingår i: Small Angle Scattering Part B: Methods for Structural Interpretation. - : Elsevier. - 0076-6879 .- 1557-7988. - 9780323991810 ; 678, s. 299-330
-
Bokkapitel (refereegranskat)abstract
- Intrinsically disordered proteins (IDPs) have a broad energy landscape and consequently sample many different conformations in solution. The innate flexibility of IDPs is exploited in their biological function, and in many instances allows a single IDP to regulate a range of processes in vivo. Due to their highly flexible nature, characterizing the structural properties of IDPs is not straightforward. Often solution-based methods such as Nuclear Magnetic Resonance (NMR), Förster Resonance Energy Transfer (FRET), and Small-Angle X-ray Scattering (SAXS) are used. SAXS is indeed a powerful technique to study the structural and conformational properties of IDPs in solution, and from the obtained SAXS spectra, information about the average size, shape, and extent of oligomerization can be determined. In this chapter, we will introduce model-free methods that can be used to interpret SAXS data and introduce methods that can be used to interpret SAXS data beyond analytical models, for example, by using atomistic and different levels of coarse-grained models in combination with molecular dynamics (MD) and Monte Carlo simulations.
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| 35. |
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| 36. |
- Li, Y, et al.
(författare)
-
Yeast RNA polymerase II holoenzyme.
- 1996
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Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 273, s. 172-5
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Tidskriftsartikel (refereegranskat)
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| 37. |
- Liu, Quanli, 1988, et al.
(författare)
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Modular Pathway Rewiring of Yeast for Amino Acid Production
- 2018
-
Ingår i: Methods in Enzymology. - : Elsevier. - 1557-7988 .- 0076-6879. ; 608, s. 417-439
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Amino acids find various applications in biotechnology in view of their importance in the food, feed, pharmaceutical, and personal care industries as nutrients, additives, and drugs, respectively. For the large-scale production of amino acids, microbial cell factories are widely used and the development of amino acid-producing strains has mainly focused on prokaryotes Corynebacterium glutamicum and Escherichia coli. However, the eukaryote Saccharomyces cerevisiae is becoming an even more appealing microbial host for production of amino acids and derivatives because of its superior molecular and physiological features, such as amenable to genetic engineering and high tolerance to harsh conditions. To transform S. cerevisiae into an industrial amino acid production platform, the highly coordinated and multiple layers regulation in its amino acid metabolism should be relieved and reconstituted to optimize the metabolic flux toward synthesis of target products. This chapter describes principles, strategies, and applications of modular pathway rewiring in yeast using the engineering of L-ornithine metabolism as a paradigm. Additionally, detailed protocols for in vitro module construction and CRISPR/Cas-mediated pathway assembly are provided.
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| 38. |
- Logan, Derek T., et al.
(författare)
-
Interactive model building in neutron macromolecular crystallography
- 2020
-
Ingår i: Methods in Enzymology. - : Elsevier. - 1557-7988 .- 0076-6879. - 9780128192146 ; 634, s. 201-224
-
Bokkapitel (refereegranskat)abstract
- This chapter aims to give an overview of the process of interactive model building in macromolecular neutron crystallography for the researcher transitioning from X-ray crystallography alone. The two most popular programs for refinement and model building, phenix.refine and Coot, respectively, are used as examples, and familiarity with the programs is assumed. Some work-arounds currently required for proper communication between the programs are described. We also discuss the appearance of nuclear density maps and how this differs from that of electron density maps. Advice is given to facilitate deposition of jointly refined neutron/X-ray structures in the Protein Data Bank.
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| 39. |
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| 40. |
- Lundmark, Richard, et al.
(författare)
-
Expression and properties of sorting nexin 9 in dynamin-mediated endocytosis
- 2005
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Ingår i: Methods in Enzymology. - : Elsevier. - 0076-6879 .- 1557-7988. ; 404, s. 545-556
-
Tidskriftsartikel (refereegranskat)abstract
- Sorting nexin 9 (SNX9) is identified as an important regulator of dynamin function in clathrin-mediated endocytosis. SNX9 recruits dynamin to the plasma membrane and promotes its GTPase activity, resulting in membrane constriction and ultimate transport vesicle scission. This chapter describes procedures to express recombinant SNX9, to biochemically characterize the cytosolic complex between SNX9 and dynamin, and to identify additional interacting partners of SNX9. Assays are presented to investigate the requirements for SNX9-dependent membrane recruitment of dynamin in vitro and in vivo.
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| 41. |
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| 42. |
- Nissan, Tracy, et al.
(författare)
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Analyzing P-bodies in Saccharomyces cerevisiae
- 2008
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Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 448, s. 507-520
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Tidskriftsartikel (refereegranskat)abstract
- Cytoplasmic processing bodies, or P-bodies, are RNA-protein granules found in eukaryotic cells. P-bodies contain non-translating mRNAs and proteins involved in mRNA degradation and translational repression. P-bodies, and the mRNPs within them, have been implicated in mRNA storage, mRNA degradation, and translational repression. The analysis of mRNA turnover often involves the analysis of P-bodies. In this chapter, we describe methods to analyze P-bodies in the budding yeast, Saccharomyces cerevisiae, including procedures to determine whether a protein or mRNA can accumulate in P-bodies, whether an environmental perturbation or mutation affects P-body size and number, and methods to quantify P-bodies.
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| 43. |
- Ohlson, Johan, et al.
(författare)
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A method for finding sites of selective adenosine deamination
- 2007
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Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 424, s. 289-300
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Forskningsöversikt (refereegranskat)abstract
- Single sites of selective adenosine (A) to inosine (1) RNA editing with functional consequences on the proteome are rarely found in mammals. Here we describe a method that can be used to detect novel site-selective A-to-I editing in various tissues as well as species. The method utilizes immunoprecipitation of intrinsic RNA-protein complexes to extract substrates subjected to site-selective in vivo editing. We show that known single sites of A-to-I editing are enriched utilizing an antibody against the ADAR2 protein. We propose that this method is suitable for identification of novel substrates subjected to site-selective A-to-I editing.
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| 44. |
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| 45. |
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| 46. |
- Royce, Thomas E., et al.
(författare)
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Extrapolating traditional DNA microarray statistics to tiling and protein microarray technologies
- 2006
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Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 411, s. 282-311
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Tidskriftsartikel (refereegranskat)abstract
- A credit to microarray technology is its broad application. Two experiments--the tiling microarray experiment and the protein microarray experiment--are exemplars of the versatility of the microarrays. With the technology's expanding list of uses, the corresponding bioinformatics must evolve in step. There currently exists a rich literature developing statistical techniques for analyzing traditional gene-centric DNA microarrays, so the first challenge in analyzing the advanced technologies is to identify which of the existing statistical protocols are relevant and where and when revised methods are needed. A second challenge is making these often very technical ideas accessible to the broader microarray community. The aim of this chapter is to present some of the most widely used statistical techniques for normalizing and scoring traditional microarray data and indicate their potential utility for analyzing the newer protein and tiling microarray experiments. In so doing, we will assume little or no prior training in statistics of the reader. Areas covered include background correction, intensity normalization, spatial normalization, and the testing of statistical significance.
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| 47. |
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| 48. |
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| 49. |
- Scheele, Camilla, et al.
(författare)
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USING FUNCTIONAL GENOMICS TO STUDY PINK1 AND METABOLIC PHYSIOLOGY :
- 2009
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Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 457, s. 211-229
-
Forskningsöversikt (refereegranskat)abstract
- Genome sequencing projects have provided the substrate for an unimaginable number of biological experiments. Further, genomic technologies such as microarrays and quantitative and exquisitely sensitive techniques such as real-time quantitative polymerase chain reaction have made it possible to reliably generate millions of data points per experiment. The data can be high quality and yield entirely new insights into how gene expression is coordinated under complex physiological situations. It can also be that the data and interpretation are meaningless because of a lack of physiological context or experimental control. Thus, functional genomics is now being applied to study metabolic physiology with varying degrees of success. From the genome sequencing projects we also have the information needed to design chemical tools that can knock down a gene transcript, even distinguishing between splice variants in mammalian cells. Use of such technologies, inspired by nature's endogenous RNAi mechanism-microRNA targeting, comes with significant caveats. While the discipline of Pharmacology taught us last century that inhibitor action specificity is dependent on the concentration used, these experiences have been ignored by users of siRNA technologies. What we provide in this chapter is some considerations and observations from functional genomic studies. We are largely concerned with the phase that follows a microarray study, where a candidate gene is selected for manipulation in a system that is considered to be simpler than the in vivo mammalian tissue and thus the methods discussed largely apply to this cell biology phase. We apologize for not referring to all relevant publications and for any technical considerations we have also failed to factor into our discussion.
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| 50. |
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