Hptlc 5651 plate

High-performance thin-layer chromatography (HPTLC) analysis of the gangliosides was performed using a 10 × 10 cm HPTLC 5651 plate (Merck, Rahway, NJ, USA), as previously described [68 (link)]. Purified gangliosides (100 μg of protein/50 μL/lane) were separated using the HPTLC plates, which were subsequently developed using chloroform/MeOH/0.25% CaCl₂·H₂O (50:40:10, v/v/v). Gangliosides were visualized via 0.2% resorcinol staining and then the HPTLC plates were dried at 105 °C in a drying oven for at least 3 h. Monosialoganglioside (Matreya LLC, State College, PA, USA), disialoganglioside (Matreya LLC, State College, PA, USA), and rat brain gangliosides were used as markers for individual ganglioside species.

Gangliosides extraction and purification were performed using DAEA Sephadex A25 column and Sep-Pak C18 cartridge, as previously described [14 (link)]. Also, HPTLC analysis of the gangliosides was performed using a 10 × 10 cm HPTLC 5651 plate (Merck, Frankfurter, Darmstadt, Germany), as previously described [14 (link)]. The purified gangliosides were normalized by total protein concentration and were loaded (500 μg protein/lane) onto TLC 5651 plates that were subsequently developed in chloroform/methanol/0.25% CaCl₂·H₂O (50:40:10, v/v/v) and visualized using 0.2% resorcinol. Gangliosides from murine and bovine brains were used as markers for individual ganglioside species.