Blotting of glycolipids and phospholipids from a high-performance thin-layer chromatogram to a polyvinylidene...

A simple method of blotting glycosphingolipids from a high-performance thin-layer chromatography (HPTLC) plate to a polyvinylidene difluoride (PVDF) membrane is described. The developed HPTLC plate is dipped in a solvent mixture (isopropanol/0.2% CaCl2/methanol, 40/20/7 by volume) for blotting, after which first a PVDF membrane and then a glass microfiber filter is placed on the plate. The assemblage then is pressed for 30 s with heating. Most of the glycosphingolipids that are separated on the HPTLC plate can be blotted quantitatively and detected with the reagents used on the plate. Detection of the glycosphingolipids on the membrane was confirmed to be more sensitive than that on the HPTLC plate by both chemical visualization and immunological staining. The glycosphingolipids blotted on the membrane could be reextracted. Blotting of phospholipids can be done by the same method. The results suggest that this method can be used in the purification and characterization of glycosphingolipids and in the detection of proteins which recognize glycosphingolipids and phospholipids.