α2β1-integrin Clustering and Internalization Protocol

Abstract

α2β1-integrin clustering experiment can be used to trigger internalization of α2β1-integrin. When clustering is performed with sequential administration of primary and fluorescent secondary antibodies, the entry kinetics of integrin can be followed into the cell. The idea is first to allow binding of primary antibodies (recognizing the extracellular epitope) to the α2β1-integrins and then to cluster the α2β1-integrin-bound primary antibodies together by the means of the secondary antibody. Binding is done on ice so that the α2β1-integrins will not internalize before both sets of antibodies are bound. Clustering is known to trigger α2β1-integrin internalization efficiently from the cell surface to the cytoplasm. In this protocol we used antibody-induced clustering of α2β1-integrin in order to quantitate the amount of internalized α2β1-integrins in comparison to cell surface-associated α2β1-integrin.