Expression and purification of the mammalian translocator protein for structural studies
(A) Overview of the purification steps. (B) Coomassie blue stained SDS-PAGE gel and in gel fluorescence of protein at various stages of purification. The lanes are labelled as follows: “M”—molecular weight marker, “SN”—supernatant, “FT” flow-through, “W1”—wash 1, “W2”—wash 2, “elu”–IMAC elution, “des”—desalting, “3C”–protein after 3C cleavage overnight, “rIMAC”—flow-through of the reverse IMAC procedure, “SEC”—pooled fractions after SEC. The final sample after SEC is monodisperse and does not contain any YFP. (C) Final construct after purification. (D) Size-exclusion profile of cleavable pig TSPO. The fractions of the peak taken for subsequent studies are indicated with the dashed lines.