Clathrin and AP fractions were purified as previously described (Campbell et al., 1984 (link); Ahle and Ungewickell, 1986 (link); Wu et al., 2010 (link)) from the coat fraction by gel filtration through a Tricon 10/300 Superose 6 column (GE Healthcare Life Sciences). Fractions containing clathrin or AP fraction (primarily consisting of AP2, but also containing small percentages of AP180 [Ahle and Ungewickell, 1986 (link)], AP1 [Keen, 1987 (link)], and Auxilin [Ahle and Ungewickell, 1990 (link)]) were pooled separately, dialyzed, and concentrated before being flash frozen in liquid nitrogen and stored at −80°C.
Purified clathrin was conjugated with Alexa fluorophores as described previously (Wu et al., 2010 (link)). Alexa Fluor 555 maleimide (Invitrogen A20346) and Alexa Fluor 647 maleimide (Invitrogen A20347) were used to label the purified clathrin. Conjugated clathrin were subjected to a round of assembly and disassembly to ensure functionality. The protein and dye concentrations were estimated from the absorbance at 280 nm, 555 nm, or 647 nm, respectively. The ratio of dye to protein was then calculated for each batch; values were typically in the range of 2.5–3.5 dyes per protein molecule.
Purified clathrin was conjugated with Alexa fluorophores as described previously (Wu et al., 2010 (link)). Alexa Fluor 555 maleimide (Invitrogen A20346) and Alexa Fluor 647 maleimide (Invitrogen A20347) were used to label the purified clathrin. Conjugated clathrin were subjected to a round of assembly and disassembly to ensure functionality. The protein and dye concentrations were estimated from the absorbance at 280 nm, 555 nm, or 647 nm, respectively. The ratio of dye to protein was then calculated for each batch; values were typically in the range of 2.5–3.5 dyes per protein molecule.
Free full text: Click here
