Gangliosides and Membrane Microdomains

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling via plasma membrane. Elucidating the precise distribution of GM3 helps to understand its function. Although several anti-GM3 monoclonal antibodies are established, the results from the immunostaining of the antibody on the cell surface need to be carefully evaluated: the antibody binds to GM3 in sparse but not in confluent cells, and the binding is temperature dependent. Our model membrane study evidenced that GM3 makes clusters in the less fluid membrane environment, where monoclonal anti-GM3 antibodies showed stronger binding. These results suggest that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 clusters differ in different membrane environments. This chapter describes the detailed protocols to examine the binding of anti-GM3 antibodies to different lipid mixtures by solid phase reaction, as well as to high- and low-density melanoma cells. This chapter also describes the methods to study the clustering of fluorescent GM3 analogs in different lipid compositions at various temperatures.