Journal:

Article Title: Purification, cloning and characterization of a GPI inositol deacylase from Trypanosoma brucei

doi: 10.1093/emboj/20.17.4923

Figure Lengend Snippet: Fig. 3. Purification and microsequencing of the inositol deacylase. [3H]DFP-labelled bloodstream-form T.brucei membranes (4 × 1011 cell equivalents) were washed with high-pH buffer and extracted with nOG. The soluble fraction (lane 1) was adsorbed with anti-VSG–Sepharose and the supernatant (lane 2) was adsorbed with ConA–agarose. The unbound material (lane 3) was discarded and the α-methyl-mannoside eluate (lane 4) was fractionated by TMAE–Fractogel FPLC. The radioactive fractions eluting at 400 mM NaCl (lane 5) were diafiltered and applied to a hydroxyapatite FPLC column. An aliquot (1%) of the radioactive fraction eluting at 200 mM sodium phosphate (lane 6) was analysed by SDS–PAGE and fluorography (lane 7). The silver-stained gel (lanes 1–6) represents 0.0005% (lanes 1–3) or 1% (lanes 4–6) of the total material. A preparative gel of the material shown in lanes 6 and 7 was used to excise the band indicated by the arrow for in-gel trypsin digestion. The tryptic peptides were fractionated by capillary microbore HPLC and the Edman sequencing results of five peptides are indicated. Residues in parentheses were ambiguous.

Article Snippet: This material was applied to a TMAE–Fractogel FPLC column (1.5 × 15 cm; Merck) pre-equilibrated with buffer D (20 mM Tris–HCl pH 7.2, 0.1% nOG, 50 mM NaCl, 2 mM EDTA, 0.25 M α-methyl-mannoside).

Techniques: Purification, SDS Page, Staining, Sequencing