Figure 6. Analysis and identification of caveolae proteins by surface biotinylation, one- and two-dimensional gel electrophoresis.

(A) Caveolae were immunoseparated from DRM and bound (B) and unbound (UB) fractions were resolved by SDS/PAGE and silver-stained. The gel lanes were sliced into equally sized pieces and analysed by MS/MS. Proteins identified on the basis of MS/MS peptide sequences from the bound fraction are listed alongside the corresponding gel piece. (B) Labelled caveolae proteins as visualized by biotin detection after immunoblotting of immunoseparated proteins (bound fraction) from DRM fractions, isolated from surface-biotinylated HUVEC, clearly showing the abundant presence of CD59 (band at 18 kDa). (C) Part of a silver-stained two-dimensional gel representing captured caveolae proteins, revealing differently shaped and sized vimentin spots. Identified proteins, molecular masses and pI markers are indicated.