particularly the aggregation status and homogeneity of the MPs are
evaluated by different methods. Native-PAGE helps to ensure that
the protein can migrate inside of the gel, suggesting a
non-aggregation state (Fig. 3a). This is further confirmed by size
exclusion chromatography (SEC, Fig. 3c) which do not show any
protein signal at the void volume. The protein homogeneity and
oligomeric state can also be investigated by SEC, Native-PAGE,
and chemical cross-linking (Fig. 3b).
2 Materials
All solutions are prepared using ultrapure water (18.2 MΩ-cm
conductivity at 25
C) and analytical grade reagents. All chemicals
are obtained from Sigma-Aldrich (unless indicated otherwise).
2.1 Solubilization
Screening
1. Bio-Dot
® microfiltration apparatus (Bio-Rad).
2. Phosphate-buffered saline (1Â PBS): 25 mM Na 2 HPO 4 ,
150 mM NaCl, pH 8.0.
Fig. 2 Membrane protein stability assessment. (a) Western blot based thermostability assay (adapted from
[12]). (b) Functional investigation using radioligand binding and ATPase activity assays for a GPCR and a
Transporter target, respectively. Two examples were previously described [9, 11]
Solubilization and Stabilization of Membrane Proteins
259