A Step-by-Step Guide to Single-Subunit Counting of Membrane-Bound Proteins in Mammalian Cells

Determining the composition and stoichiometry of membrane-bound proteins has been a perennial problem that has plagued biology for a long time. The most recurring issue is that composition and subunit stoichiometry is commonly inferred from bulk biochemical assays that can only shed light on the “averaged” makeup of the protein complex. However, recent studies have been able to circumvent this issue by studying the stoichiometry of individual protein complexes. The most common approach has been to express GFP-tagged subunits in Xenopus laevis oocytes and then manually count the number of photobleaching steps to report mature protein stoichiometry. Although valuable, an important drawback of this technique is that the strict rules of mammalian protein assembly are not always adhered to in this surrogate expression system. Furthermore, manual counting of bleaching steps is subject to user bias and places practical limits on the amount of data that can be analyzed. In this chapter, we provide a step-by-step account of how we adapted the subunit counting method for mammalian cells to study the composition and stoichiometry of ionotropic glutamate receptors. Using custom-made software, we have automated the entire counting process so that it is much less time consuming and no longer subject to user bias. Given its universality, this methodological approach permits the elucidation of subunit number and stoichiometry for a wide variety of plasma-membrane-bound proteins i...
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