Isolation of total polysaccharides from H. pallida (A) and C. intestinalis (B). A and B, the polysaccharides extracted from ascidians (~15 mg) before (-) and after (+) incubation with chondroitin AC or ABC lyases and treatment with nitrous acid, were applied to a 0.5% agarose gel and run for 1 h at 100 V in 0.05 M 1,3-diaminopropane/acetate (pH 9.0). The polysaccharides in the gel were fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solution. After 12 h, the gel was dried and stained with 0.1% toluidine blue in acetic acid/ethanol/water (0.1:5:5, v/v). As a standard, a mixture of mammalian glycosaminoglycans containing 10 mg of each chondroitin 4-sulfate (CS), dermatan sulfate (DS) and heparin (Hep) was applied in agarose gel.