GFP-tagged caveolin WT and truncation mutants were expressed (75 µl each) in the presence of a lipid dye, DiIC16 (D384; Invitrogen; at 1:1,000 dilution), and mixed with an equal volume of 80% (wt/vol) Histodenz (Sigma-Aldrich) solution in PBS. The mixed samples were loaded at the bottom of 11 × 22 mm Beckman centrifuge tubes (Beckman Instrument). The tube was sequentially overlaid with a linear Histodenz gradient (7.5%–37.5% wt/vol) in PBS. Gradients were spun at 150,000 g for 16 h in a TLS-55 rotor (Beckman Instrument) at 4°C. After that, samples were collected from the top in 50-µl increments and loaded into 384-well white plates. Fluorescence levels of GFP and DiIC16 for each fraction were read on a plate reader (Synergy 4; BioTek; excitation 485 nm and emission 520 nm for GFP and 549 nm/575 nm for DiIC16, respectively).
Jung W., Sierecki E., Bastiani M., O’Carroll A., Alexandrov K., Rae J., Johnston W., Hunter D.J., Ferguson C., Gambin Y., Ariotti N, & Parton R.G. (2018). Cell-free formation and interactome analysis of caveolae. The Journal of Cell Biology, 217(6), 2141-2165.
+ Open protocol