Protocol abstract
Nature Protocols 3, - 1782 - 1795 (2008)
Published online: 30 October 2008 | doi:10.1038/nprot.2008.177
Subject Categories: Biochemistry and protein analysis | Isolation purification and separation | Spectroscopy and structural analysis
Gel chromatography and analytical ultracentrifugation to determine the extent of detergent binding and aggregation, and Stokes radius of membrane proteins using sarcoplasmic reticulum Ca2+–ATPase as an example
Marc le Maire1,2,3, Bertrand Arnou1,2,3, Claus Olesen4, Dominique Georgin1, Christine Ebel5,6,7 & Jesper V Møller4
Abstract
For structural studies of integral membrane proteins, including their 3D crystallization, the judicious use of detergent for solubilization and purification is required. Detergent binding by the solubilized protein is an important parameter to determine the hydrodynamic properties in terms of size and aggregational (monomeric/oligo(proto)meric) state of the protein. Detergent binding can be measured by gel filtration chromatography under equilibrium conditions and after separation from mixed micelles of solubilized lipid and detergent. Using sarcoplasmic reticulum Ca2+-ATPase as an example, we demonstrate in this protocol complete procedures for measurement of detergent binding using (i) radiolabeled n-dodecyl-
-D-maltoside (DM) or (ii) from measurements of the increase in refractive index due to the presence of bound detergent on the protein. The latter measurement can also be performed by sedimentation velocity (SV) analysis in the analytical ultracentrifuge which in addition allows determination of the sedimentation coefficient. In combination with estimation of Stokes radius by gel filtration calibration, the molecular mass and asymmetry of the solubilized protein can be calculated. In the proposed protocols, the gel chromatographic procedures require 1 d; SV experiments are performed just after size exclusion. The whole time for these experiments is 24 h. Data analysis of analytical ultracentrifugation requires a couple of days.
- CEA, Institut de Biologie et Technologies de Saclay, F-91191 Gif-sur-Yvette, France.
- CNRS, Unité de Recherche Associée 2096, F-91191 Gif-sur-Yvette, France.
- Université Paris-Sud 11, LRA 17V, F-91191 Gif-sur-Yvette, France.
- Centre for Membrane Pumps in Cells and Disease—PUMPKIN, Danish National Research Foundation, Institute of Physiology and Biophysics, University of Aarhus, DK-8000 Aarhus C, Denmark.
- CEA, Direction des Sciences du Vivant, Institut de Biologie Structurale, F-38027 Grenoble, France.
- CNRS, Institut de Biologie Structurale, Unite Mixte de Recherche 5075, F-38027 Grenoble, France.
- Université Joseph Fourier, Institut de Biologie Structurale, F-38027 Grenoble, France.
Correspondence to: Marc le Maire1,2,3 e-mail: marc.lemaire@cea.fr
nature-products
A-Z product listing
- N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid(Sigma)
- 2-channel Epon centerpieces of 12-mm optical path length(Beckman)
- AUC cell assemblies equipped with sapphire windows(Beckman)
- CaCl2 (Merck)
- DM, ANAGRADE(ANATRACE Inc.)
- Folin-Ciocalteu reagent(Sigma)
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