Protein-protein contacts in solubilized membrane proteins, as detected by cross-linking.
The amount of detergent required for the solubilization of membrane proteins needs to be optimised as an excess may cause loss of activity and insufficiency may result in poor solubilization or heterogeneous samples. With sarcoplasmic reticulum Ca2+ -ATPase as an example we show by cross-linking that it can be misleading to choose the proper amount of detergent based on clarification of membrane suspensions, because clarification -as detected by turbidity measurements, for instance- precedes full protein solubilization as monomers. We demonstrate that to assess the extent of sample homogeneity at a given detergent/protein ratio, cross-linking followed by HPLC gel filtration in detergent usefully complements cross-linking followed by SDS-PAGE.
Le Maire
Marc
M
CEA, DSV, DBJC, SBFM, LRA17V, Université Paris-Sud, and CNRS, URA 2096, CEA Saclay, Gif sur Yvette F-91191, France. marc.lemaire@cea.fr
Møller
Jesper V
JV
Menguy
Thierry
T
Velours
Jean
J
Champeil
Philippe
P
D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D015203:Reproducibility of Results; D012519:Sarcoplasmic Reticulum / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility
D003432:Cross-Linking Reagents; D003902:Detergents; D008565:Membrane Proteins; D000252:Calcium-Transporting ATPases