Abstract
This protocol aims at the biotin labeling and affinity purification of plasma membrane proteins from cultured neurons. Protein biotinylation consists in the covalent attachment of biotin to proteins. Biotin is a membrane unpermeable molecule with a small size (MW 244.31 g/mol) and therefore does not interfere with the normal function of proteins. Biotin binds to streptavidin and avidin molecules with high affinity. This binding is extremely resistant to temperature, pH and proteolysis, which allows capture and purification of plasma membrane proteins. Moreover, proteins can bind several biotin molecules, that will allow the consequent binding of several streptavidin or avidin molecules, increasing the sensitivity of detection of the proteins of interest. In this protocol proteins at the cell surface of live cultured neurons are biotinylated. Neuronal extracts are prepared and biotinylated proteins are collected with NeutrAvidin-coupled beads, and analyzed by Western blotting.
Keywords: Plasma-membrane proteins, Affinity purification, Biotin, Cell surface, Cultured neurons
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
Work in the authors' laboratories was supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal, and Fundo Europeu de Desenvolvimento Regional (FEDER and COMPETE).
References
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