A simple assay method for bacterial binding to glycosphingolipids on a polyvinylidene difluoride membrane after thin-layer chromatography blotting and in situ mass spectrometric analysis of the ligands.
A simple assay method for bacterial binding to glycosphingolipids on a polyvinylidene difluoride (PVDF) membrane has been developed. Glycosphingolipids were separated on a high-performance thin-layer chromatography (HPTLC) plate and transferred onto a PVDF membrane by TLC blotting [Taki, T., Handa, S., and Ishikawa, D. (1994) Anal. Biochem. 221, 312-316]. The PVDF membrane was blocked with phosphate-buffered saline containing 4% casein and 0.1% methionine and overlaid with 35S-labeled Escherichia coli possessing K99 fimbriae (E. coli K99) at 37 degrees C for more than 2 h. Binding of the 35S-labeled E. coli K99 was detected with a bioimaging analyzer. Radioactivities were located on the bands corresponding to N-glycolylneuraminic acid containing glycosphingolipids such as sialylparagloboside, GM2, and GM3 with hydroxy fatty acid in ceramide moiety, and a weak binding was detected on the band of N-acetylneuraminylparagloboside. Furthermore, an in situ mass spectrometric analysis of the ligand glycosphingolipids on the membrane was demonstrated. The present method has several advantages compared with the overlay binding assay on the HPTLC plate as follows: (i) the method is simple and rapid; (ii) the membrane is easy to handle; (iii) binding is clear with low background; (iv) a small amount of [35S]methionine is required; and (v) sensitive ligand characterization can be done by in situ mass spectrometric analysis.
Isobe
T
T
Department of Biochemistry, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113, Japan.
Naiki
M
M
Handa
S
S
Taki
T
T
D001422:Bacterial Adhesion; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer / Q000379:methods*; D004926:Escherichia coli / Q000472:pathogenicity*; D006028:Glycosphingolipids / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D011145:Polyvinyls; D005465:Fluorocarbon Polymers
D006028:Glycosphingolipids; D008024:Ligands; D008567:Membranes, Artificial; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers