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P-Type ATPases

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This volume provides a broad collection of protocols for many of the common experimental techniques used for the characterization of P-type ATPases. P-Type ATPases: Methods and Protocols provides comprehensive practical instructions for all researchers in the P-type ATPase field, from the protein biochemist to the mouse geneticist, covering the identification and classification, isolation, purification, in vitro characterization, knock-out studies, as well as crystallization and structural analysis. Written for the Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and practical, P-Type ATPases: Methods and Protocols aims to ensure successful results in the further study of this vital field of research.


1.      An Introduction to P-type ATPase research

 Poul Nissen

Part I: Protein Production, Isolation, Purification, and Stabilization

2.      Purification of Na,K-ATPase from Pig Kidney

 Natalya U. Fedosova

3.      Preparation of Ca2+-ATPase1a Enzyme from Rabbit Sarcoplasmic Reticulum

 Jesper V. Møller and Claus Olesen

4.      Isolation of H+/K+-ATPase-enriched Membrane Fraction from Pig Stomachs

 Kazuhiro Abe and Claus Olesen

5.      Overproduction of PiB-type ATPases

Xiangyu Liu, Oleg Sitsel, Kaituo Wang, and Pontus Gourdon

6.      Coordinated Overexpression in Yeast of a P4-ATPase and its Associated Cdc50 Subunit: The Case of the Drs2p/Cdc50p Lipid Flippase Complex

 Hassina Azouaoui, Cédric Montigny, Aurore Jacquot, Raphaëlle Barry, Philippe Champeil, and Guillaume Lenoir

7.      The Plasma Membrane Ca2+-ATPase: Purification by Calmodulin Affinity Chromatography, and Reconstitution of the Purified Protein

Verena Niggli and Ernesto Carafoli

8.      Expression of Na,K-ATPase and H,K-ATPase Isoforms With The Baculovirus Expression System

 Jan B. Koenderink and Herman G.P. Swarts

9.      Time-dependent Protein Thermostability Assay

 Ilse Vandecaetsbeek and Peter Vangheluwe


Part II : Activity Assays

10.  Colorimetric Assays of Na,K-ATPase

 Kathleen J. Sweadner

11.  ATPase Activity Measurements by an Enzyme-Coupled Spectrophotometric Assay Pankaj Sehgal, Claus Olesen, and Jesper V. Møller

12.  Antimony-Phosphomolybdate ATPase Assay

 Gianluca Bartolommei and Francesco Tadini-Buoninsegni

13.  ATPase Activity Measurements using radiolabeled ATP

Herman G.P. Swarts and Jan B. Koenderink

14.  Assaying P-type ATPases Reconstituted in Liposomes

 Hans-Jürgen Apell and Bojana Damnjanovic

15.  Coupling ratio for Ca2+ Transport by Calcium Oxalate Precipitation

Pankaj Sehgal, Claus Olesen, and Jesper V. Møller

16.  Calcium Uptake in Crude Tissue Preparation

 Philip A. Bidwell and Evangelia G. Kranias

17.  Measuring H+ Pumping and Membrane Potential Formation in Sealed Membrane Vesicle Systems

Alex Green Wielandt, Michael G. Palmgren, Anja Thoe Fuglsang, Thomas Günther Pomorski, and Bo Højen Justesen

18.  Assay of Flippase Activity in Proteoliposomes using Fluorescent Lipid Derivatives Magdalena Marek and Thomas Günther Pomorski


Part III : In vitro Functional Studies

19.  The use of Metal Fluoride Compounds as Phosphate Analogs for Understanding the Structural Mechanism in P-type ATPases

 Stefania J. Danko and Hiroshi Suzuki

20.  Phosphorylation/Dephosphorylation Assays

Hiroshi Suzuki

21.  Tryptophan Fluorescence Changes Related to Ca2+-ATPase Function

Pankaj Sehgal, Claus Olesen, and Jesper V. Møller


Part IV: Ligand Binding Studies

22.  Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca2+-ATPase by Competitive Inhibition of [g-32P]TNP-8N3-ATP Photolabeling

 Johannes D. Clausen, David B. McIntosh, David G. Woolley, and Jens Peter Andersen

23.  Ca2+ Binding and Transport Studied with Ca2+/EGTA Buffers and 45Ca2+

 Pankaj Sehgal, Claus Olesen, and Jesper V. Møller

24.  Assay of Copper Transfer and Binding to P1B-ATPases

 Teresita Padilla-Benavides and José M. Argüello


Part V: Electrophysiology

25.  Voltage Clamp Fluorometry of P-Type ATPases

 Robert E. Dempski

26.  Electrophysiological Measurements on Solid Supported Membranes

Francesco Tadini-Buoninsegni and Gianluca Bartolommei

27.  Electrophysiological Characterization of Na,K-ATPases Expressed in Xenopus laevis Oocytes Using Two-Electrode Voltage Clamping

 Florian Hilbers and Hanne Poulsen


Part VI: Functional Studies by Cell Culture and Transgenic Animals

28.  Functional Studies of Na+,K+-ATPase using Transfected Cell Cultures

 Elena Arystarkhova and Kathleen J. Sweadner

29.  HPLC Neurotransmitter Analysis

 Thomas Hellesøe Holm, Toke Jost Isaksen, and Karin Lykke-Hartmann

30.  Behavior Test Relevant to a2/a3Na+/K+-ATPase Gene Modified Mouse Models

 Toke Jost Isaksen, Thomas Hellesøe Holm, and Karin Lykke-Hartmann

31.  Zebrafish Whole-mount in situ Hybridization Followed by Sectioning

 Canan Doganli, Jens Randel Nyengaard, and Karin Lykke-Hartmann

32.  Whole-mount Immunohistochemistry for Anti-F59 in Zebrafish Embryos (1-5 days Post Fertilization (dpf)) Canan Doganli

 Lucas Bukata and Karin Lykke-Hartmann

33.  Cell-based Lipid Flippase Assay Employing Fluorescent Lipid Derivatives

 Maria S. Jensen, Sara Costa, Thomas Günther-Pomorski, and Rosa L. López-Marqués

34.  Transient Expression of P-type ATPases in Tobacco Epidermal Cells

 Lisbeth R. Poulsen, Michael G. Palmgren, and Rosa L. López-Marqués 


Part VII: Lipid Techniques

35.  Lipid Exchange by Ultracentrifugation

Nikolaj D. Drachmann and Claus Olesen

36.  Reconstitution of Na+, K+-ATPase in Nanodiscs

Jonas Lindholt Gregersen, Natalya Fedosova, Poul Nissen, and Thomas Boesen

Part VIII: Crystallization

37.  Crystallization of P-type ATPases by the High Lipid-detergent (HiLiDe) Method

 Oleg Sitsel, Kaituo Wang, Xiangyu Liu, and Pontus Gourdon

38.  Two-dimensional Crystallization of the Ca2+-ATPase for Electron Crystallography John Paul Glaves, Joseph O. Primeau, and Howard S. Young

39.  Two-dimensional  Crystallization  of  Gastric  H+,K+-ATPase  for  Structural  Analysis  By Electron Crystallography

 Kazuhiro Abe

Part IX: Computational Approaches in analyzing P-type ATPases

40.  MD Simulations of P-type ATPases in a Lipid Bilayer System

 Henriette Elisabeth Autzen and Maria Musgaard

41.  Computational Classification of P-Type ATPases

 Dan Søndergaard, Michael Knudsen, and Christian Nørgaard Storm Pedersen

42.  Molecular Modeling of Fluorescent SERCA Biosensors

 Bengt Svensson, Joseph M. Autry, and David D. Thomas

43.  How to Compare, Analyze and Morph Between Crystal Structures of Different Conformations: The P-type ATPase Example

Jesper L. Karlsen and Maike Bublitz


Titel: P-Type ATPases
Untertitel: Methods and Protocols
EAN: 9781493931781
ISBN: 978-1-4939-3178-1
Format: Fester Einband
Herausgeber: Springer, Berlin
Genre: Biologie
Anzahl Seiten: 544
Gewicht: g
Größe: H39mm x B260mm x T186mm
Jahr: 2015
Auflage: 1st ed. 2016

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