Metabolic Radiolabeling of Animal Cell Glycoconjugates

Abstract

This unit describes metabolic labeling techniques that provide specific information about the structure, sequence, and distribution of the sugar chains of glycoconjugates. Although these techniques provide less information than complete sequencing of the sugar chains, the partial structural information derived is sufficient for many purposes. In the basic procedure presented in this unit, actively growing cell cultures are grown through several population doublings in complete medium supplemented with radiolabeled glycoconjugate precursors to reach a steady-state level of incorporation. In alternate protocols, cells are cultured for a short period of time in a deficient medium that contains a high concentration of radiolabeled precursor. A pulse or pulse-chase labeling procedure can be used to analyze precursor-product relationships. With sequential pulse-labeling, it is possible to obtain quantities of labeled glycoconjugates with the use of a minimal amount of labeled precursor by using the same medium to pulse-label a series of cultures. A support protocol describes the preparation of multiply deficient medium (MDM) for use in making appropriate deficient media.