Fig. 3

Purification and reactivity of recombinant Fab fragment from clone 65. a Purification of Fab 65 from E. coli Top10F’ crude extracts after IMAC purification and subsequent affinity purification with protein L agarose beads. Samples were separated under non-reducing conditions in a 10% polyacrylamide gel stained with coomassie brilliand blue. M: prestained protein standards (NEB, P7712). Lanes 1–3: three eluates of the last purification step from the protein L agarose beads. Arrows indicate the 46 and 58 kDa molecular mass standard positions, respectively. b Western blot. Lanes 1–2: probed with a Fab 65 E. coli Top10F’ crude extract, lanes 3–4: probed with a E. coli Top10F’ Fab TT crude extract (kindly provided by the Carlos F. Barbas III laboratory, the Scripps Research Institute), lanes 5–6: probed only with the secondary antibody. Lanes 1, 3, 4: contain 20 μg NPCs lysate, lanes 2, 4, 6: contain 20 μg mouse spinal cord lysate. Arrowheads indicate the 25, 32 and 46 kDa molecular mass standard positions, respectively