Structuring Detergents for Extracting and Stabilizing Functional Membrane Proteins
Figure 3
Extraction of membrane proteins by C4Cn derivatives.
Extraction of ABC transporters by C4Cn, prokaryotic BmrA (A) and YheI/YheH (B) expressed in E. coli, human ABCG2 expressed in Sf9 (C) or HEK293 (D) cells, together with AcrB expressed in E. coli (E) and the SR-Ca2+-ATPase (F), was carried out on the corresponding membrane fractions as described in Methods. After solubilisation, extracted and non-extracted materials were separated by high-speed centrifugation, generating a supernatant S and a pellet P which were loaded on a 10% SDS-PAGE, and after migration either stained with Coomassie blue or submitted to a Western blot for ABCG2 (upper lanes in panels in C and D). Arrows indicate the position of each monomer. Positive control experiments were carried out with DDM, FC12 and C12E8, negative controls were carried out without detergent (No det). The red dotted line indicates the threshold of extraction.