Cell surface biotinylation experiments

(A) Biotin labeling of the surface of neuronal cultures allows efficient purification of the surface only protein fraction through precipitation with NeutrAvidin beads. Modification of the protocol to include one or more cleavage steps allows labelling of specific compartments. Endocyctic compartments are labelled by returning dishes to 37C after surface labelling to allow internalisation. (B) Endocytosis can be halted again by returning the dishes to 4C, at which point remaining surface labels can be cleaved away. The protected pool of endocytosed receptors is purified after the cells are solubilised. (C) In an extension of the endocytosis protocol, the re-insertion of surface proteins can also be measured. Following the cleavage of the remaining label from the surface, returning the dishes to 37C with continued cleavage from the surface reduces the size of the protected internal pool over time.

This figure was part of my PhD thesis, completed in 2010.