Abstract
KISS (KInase Substrate Sensor) is a recently developed two-hybrid technology that allows in situ analysis of protein-protein interactions in intact mammalian cells. In this method, which is derived from MAPPIT (mammalian protein-protein interaction trap), the bait protein is coupled to the kinase domain of TYK2, while the prey protein is fused to a fragment of the gp130 cytokine receptor chain. Bait and prey interaction leads to phosphorylation of the gp130 anchor by TYK2, followed by recruitment and activation of STAT3, resulting in transcription of a STAT3-dependent reporter system. This approach enables the identification of interactions between proteins, including transmembrane and cytosolic proteins, and their modulation in response to physiological or pharmacological challenges. Here, we describe a detailed step-by-step protocol for the detection of an interaction between two proteins of interest using KISS.
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