P-Type ATPases Methods and Protocols

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.

An Introduction to P-type ATPase research.-  Purification of Na,K-ATPase from Pig Kidney.- Preparation of Ca²⁺-ATPase1a Enzyme from Rabbit Sarcoplasmic Reticulum.- Isolation of H⁺/K⁺-ATPase-enriched Membrane Fraction from Pig Stomachs.- Overproduction of PiB-type ATPases.- Coordinated Overexpression in Yeast of a P4-ATPase and its Associated Cdc50 Subunit: The Case of the Drs2p/Cdc50p Lipid Flippase Complex.- The Plasma Membrane Ca²⁺-ATPase: Purification by Calmodulin Affinity Chromatography, and Reconstitution of the Purified Protein.- Expression of Na,K-ATPase and H,K-ATPase Isoforms With The Baculovirus Expression System.- Time-dependent Protein Thermostability Assay.- Colorimetric Assays of Na,K-ATPase.- ATPase Activity Measurements by an Enzyme-Coupled Spectrophotometric Assay.- Antimony-Phosphomolybdate ATPase Assay.- ATPase Activity Measurements using radiolabeled ATP.- Assaying P-type ATPases Reconstituted in Liposomes.- Coupling ratio for Ca²⁺ Transport by Calcium Oxalate Precipitation.- Calcium Uptake in Crude Tissue Preparation.- Measuring H⁺ Pumping and Membrane Potential Formation in Sealed Membrane Vesicle Systems.- Assay of Flippase Activity in Proteoliposomes using Fluorescent Lipid Derivatives.- The use of Metal Fluoride Compounds as Phosphate Analogs for Understanding the Structural Mechanism in P-type ATPases.- Phosphorylation/Dephosphorylation Assays.- Tryptophan Fluorescence Changes Related to Ca²⁺-ATPase Function.- Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca²⁺-ATPase by Competitive Inhibition of [g-32P]TNP-8N3-ATP Photolabeling.

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

24.  Assay of Copper Transfer and Binding to P_1B-ATPases

25.  Voltage Clamp Fluorometry of P-Type ATPases

26.  Electrophysiological Measurements on Solid Supported Membranes

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

29.  HPLC Neurotransmitter Analysis

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

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

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

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

35.  Lipid Exchange by Ultracentrifugation

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

38.  Two-dimensional Crystallization of the Ca²⁺-ATPase for Electron Crystallography

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

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

41.  Computational Classification of P-Type ATPases

42.  Molecular Modeling of Fluorescent SERCA Biosensors

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

Jesper L