Fig. 1.
Analysis of PCDH-γ macromolecular protein complexes using sucrose gradient ultracentrifugation and 2D BN/SDS-PAGE. A, SG ultracentrifugation analysis of PCDH-γ complexes. Brain membrane protein extracts from P0 or P21 mice were subjected to ultracentrifugation on a 5–50% sucrose gradient. Wild-type (WT; P0) membrane proteins were also treated with 1% SDS before SG ultracentrifugation to dissociate non-covalently linked PCDH-γ complexes. Samples from Pcdh-γdel/del mice served as a negative control to show the specificity of Western blot analysis. B, 2D BN/SDS-PAGE analysis of PCDH-γ complexes. Crude membrane protein extracts were subjected to 2D BN/SDS-PAGE analysis followed by Western blot analysis using an anti-pan-PCDH-γ antibody. The NativeMark unstained protein standard (Invitrogen) was used as molecular mass standard. The arrowhead in B indicates protein aggregation at the gel entry point (well bottom). THY, thyroglobulin; BD, blue dextran.