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. 2012 Aug 22;23(1):23–31. doi: 10.1093/glycob/cws123

Fig. 1.

Fig. 1.

Affinity overlay of LT-IIc binding to commercial ganglioside standards. (A) A mixture of disialogangliosides was plated on a TLC plate and overlaid with LT-IIc. Blocking was performed with 1–3% BSA (lanes 1–3) and 2.5–5.0% non-fat dry milk (lanes 4 and 5) before being overlaid with LT-IIc. Ganglioside positions are noted on the right margin: a, GD3; b, GD1a; c, GD1b. (B) LT-IIc was overlaid on a mixture of mono- and di-sialogangliosides on TLCs at increasing concentrators (0.1–0.5 μg/mL) and affinity overlay immunoblots were made. Ganglioside positions are noted on the right margin: a, GM3; b, GM2; c, GM1a; d, GD3; e, GD1a; f, GD1b. Gangliosides were also overlaid with buffer alone (lane 4) to confirm the absence of non-specific binding. (C) LT-IIc was overlaid on a, asialoGM1; b, GM1a; c, GD1a; d, GD1b; e, GT1b. Ganglioside positions are noted on the left margin. Autoradiographs were developed as detailed in Materials and methods. Glycosphingolipid nomenclature is according to Svennerholm (Svennerholm 1963): asialoGM1 (aGM1; GgOse4Cer), GM1 (II3NeuAc-GgOse4Cer), GM3 (II3NeuAc-LacCer), GM2 (II3NeuAc-GgOse3Cer), GD3 (II3(NeuAc)2-LacCer), GD1a (IV3NeuAc,II3NeuAc-GgOse4Cer), GD1b (II3(NeuAc)2, GT1b (IV3NeuAc,II3 (NeuAc)2-GgOse4Cer).