Protein–protein contacts in solubilized membrane proteins, as detected by cross-linking

• Published in: Analytical biochemistry Vol. 362; no. 2; pp. 168 - 171
• Main Authors: le Maire, Marc, Møller, Jesper V., Menguy, Thierry, Velours, Jean, Champeil, Philippe
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-03-2007; Elsevier Masson
• Subjects: Ca 2+ ATPase; Calcium-Transporting ATPases - chemistry; Calcium-Transporting ATPases - metabolism; Cellular Biology; Chemistry Techniques, Analytical - methods; Chromatography, High Pressure Liquid - methods; Cross-linking; Cross-Linking Reagents - chemistry; Detergent; Detergents - chemistry; Electrophoresis, Polyacrylamide Gel - methods; Gel filtration HPLC; Life Sciences; Membrane protein; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Protein Binding; Reproducibility of Results; Sarcoplasmic Reticulum - chemistry; Sarcoplasmic Reticulum - metabolism; Solubility; Solubilization; Membrane protein; Gel filtration HPLC; Detergent; Solubilization; Cross-linking; Ca 2+ ATPase
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2006.11.025

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 Ca
2+-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.