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Expression and purification of the mammalian translocator protein for structural studies

Fig 7

Large-scale purification of pig TSPO-YFP fusions.

(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”–eluted fractions after affinity chromatography, “SEC”—pooled fractions after SEC. (C) Large-scale purification of TSPO-YFP fusion linker10aa in different detergents. Size-exclusion chromatography of pig TSPO fusion solubilised in DDM/CHS and subsequently purified in DDM/CHS (left), LDAO/CHS (middle) and DM/CHS (right). Peaks normalised to 25 g cells. (D) Final construct after purification. (E) Tryptophan quenching assay of TSPO-YFP with diazepam and PK11195 (n = 3). (F) Thermostability of the purified TSPO was assessed as described in “Materials and Methods”. Especially PK11195 has a stabilising effect on TSPO.

Fig 7