Isolation of Inositol 1,4,5-Trisphosphate Receptor-Associating Proteins and Selective Knockdown Using RNA Interference

Inositol 1,4,5-trisphosphate (IP₃ ) receptors (IP₃ Rs) are IP₃ -gated Ca²⁺ release channels localized on intracellular Ca²⁺ stores and play a role in the generation of complex patterns of intracellular Ca²⁺ signals. We show herein experimental protocols for the identification of associating proteins of IP₃ R isoforms from various cells and tissues using affinity column chromatography and for the specific knockdown of the expression of IP₃ R isoforms and their associating proteins using RNA interference. These methods will provide clues to understand the exact nature of how the signaling complex contributes to the generation of spatio-temporal patterns of intracellular Ca²⁺ signals.