Figure 6
Folch extraction and thin-layer chromatography of the antigen unique to the 25/45 fraction. (A) Fifteen-ÎĽl aliquots of the upper phase (U), lower phase (L), and interface (I) obtained by Folch extraction of the 25/45 fraction isolated from 20 day-old rats were separated by SDS-PAGE through 18% resolving gels and probed with MAb 10A5. Unextracted 25/45 fraction (5 ÎĽg) was included as a positive control (C). (B) Upper phase obtained by Folch extraction from 200 ÎĽg of 25/45 fraction protein was subjected to TLC through 20 Ă— 20 cm silica gel H plates and stained with resorcinol to visualize gangliosides. (C) Silica gel bands scraped from a replicate TLC plate were eluted and subjected to SDS-PAGE through 18% resolving gels. After transfer to PVDF, the membrane was immunoblotted with MAb 10A5. Note that immunoreactivity was restricted to the area of the plate containing the ganglioside (Gang). No reactivity was noted at the origin (Ori) or in other bands (B) scraped from the lane containing the upper phase. (D) Ganglioside extracted by the Folch method from the equivalent of 20, 9.7, and 6.4 ÎĽg of 25/45 fraction protein was subjected to high performance TLC (HPTLC) through 10 Ă— 10 cm silica gel 60 plates and stained with resorcinol. Bovine mixed ganglioside standards were applied at 18.8, 9.4, and 4.7 ÎĽg (per ganglioside) in alternating lanes. The arrow marks the asialo-GM1 ganglioside.