Figure 5

GnSAF immunopurified from G/LCM. Immunopurification using phage-derived antibody immobilised on protein-L agarose (a,b) or rat polyclonal antibody immobilised on anti-rat IgG-coated magnetic beads (c,d) was pooled after 15 consecutive loading and elution cycles with 2 M NaI. Proteins detected by Coomassie blue staining of (a,c) 2-D (immunopurified protein only, numbers match those in Table II) and (b, d) 1-D (Unbound = flow-through, Bound = immunopurified) gels were investigated by mass spectroscopic peptide mass mapping. Western blot of the 3-c4b-immunopurified G/LCM (e,f) with either 3-c4b and secondary antibody (e) or secondary antibody only (f) show the elution of a specifically recognised 60–70 kDa protein band (arrow) by 2 M NaI, with no further protein eluted with glycine washes.