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  • An in vitro assay that can indirectly detect the activation of small GTPases in the RAS family through the staining of phosphorylated forms of MEK (MAP2K) family proteins (pMEK). A farnesylated RAS family protein is anchored to an in vitro model of the plasma membrane and, under different conditions, is co-incubated with both a RAF family protein, which binds to and is activated by active RAS proteins, and a MEK family protein, which is phosphorylated by active RAF proteins. pMEK is detected using a donor bead coated with anti-MEK antibodies that is capable of oxidizing an acceptor molecule attached to an anti-pMEK antibody. When the donor and acceptor are in close proximity, the acceptor molecule is oxidized and can be visualized using electron microscopy.
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