P-Type ATPases, 1st ed. 2016 Methods and Protocols Methods in Molecular Biology Series, Vol. 1377

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

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 P_1B-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

 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

 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

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts