14] Detection of glycolipid ligands by direct binding of carbohydrate-binding proteins to thin-layer chromatograms

Abstract

Publisher Summary Glycolipid ligands of carbohydrate-binding proteins can be detected by binding of the I-labeled protein to thin-layer chromatograms of total lipid extracts of cells followed by autoradiography. This method, which has been used to detect glycolipid receptors for cholera toxin and monoclonal antibodies, may also be useful in defining the receptors of other proteins, such as some lectins or hormones that bind to glycolipids. Glycolipid antigens of monoclonal antibodies can be detected as follows: A total lipid extract from approximately 1 mg wet weight of tissue is chromatographed, and the chromatogram is dried and soaked in buffer A for 10 min at 4 °. The wet chromatogram was laid horizontally as before and overlayed with hybridoma culture medium containing about 10 μg of antibody per milliliter diluted 1:4 with buffer. After incubation in a humid atmosphere for 6 hr at 4 °, the chromatogram is washed by dipping in six successive changes of buffer, overlayed with 125 I-labeled F(ab') of rabbit IgG antibodies to mouse immunoglobulins in buffer A (1×10 6 cpm/ml), and incubated for an additional 12 hr in a humid atmosphere at 4 °. The chromatogram is then washed six times in buffer B, air-dried, and exposed to X-ray film.