FIG. 1.
Characterization of ST O antigen-protein conjugate. (A) Gel filtration chromatography (Bio-Gel A 0.5m) of reaction mixture containing 10 mg of HSA and 10 mg of purified ST O chains eluted with phosphate-buffered saline, pH 7.2. See Materials and Methods for details of the conjugation reaction. Contents of protein at 595 nm (▴) and carbohydrate at 480 nm (•) were determined in each fraction. The shaded area indicates fractions pooled for further analysis. Note the clear separation of conjugated material from unconjugated oxidized ST O chains. BSA, bovine serum albumin; BSA2, dimer of BSA; Ox ST O, purified oxidized ST O chains. (B) Immunoblot of 7.5% polyacrylamide gel of 2 μg of HSA, ST LPS, purified ST O chains (ST O), and ST O-HSA conjugate developed with commercial rabbit anti-ST O:1,9,12. Rabbit anti-ST O reacts with endotoxin-contaminated (Limulus amebocyte lysate assay-positive) HSA, ST LPS, and ST O-HSA conjugate. ST O did not stain because it ran out of the gel under these conditions.