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. 2000 Aug 15;19(16):4402–4411. doi: 10.1093/emboj/19.16.4402

graphic file with name cdd428f1.jpg

Fig. 1. Purification of GPI-GnT complexes. GST–FLAG-PIG-A and FLAG-HAT-PIG-A were isolated by two-step affinity purification from the digitonin lysates of 1.4 × 108 cells of JY5 transfectants. Samples of the purified proteins (equivalent to 6 × 107 cells) were used for in vitro GPI-GnT assay (A) and the rest (equivalent to 8 × 107 cells) were used for SDS–PAGE and silver staining (B). (A) Lysates derived from 107 cells of wild-type JY25 as a positive control (lane 1) and purified complexes containing GST–FLAG-PIG-A (lane 2) or FLAG-HAT-PIG-A (lane 3) were incubated with radiolabeled UDP-GlcNAc and bovine PI. Lipids were analyzed by TLC. Identities of spots are indicated on the right. (B) Silver-stained SDS–PAGE profiles of purified complexes containing GST–FLAG-PIG-A (lane 1) and FLAG-HAT-PIG-A (lane 2). Band numbers are shown on the right. The positions of the molecular size markers are indicated on the left (kDa).