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

  • Akihiro Mizutani
  • Katsuhiro Kawaai
  • Chihiro Hisatsune
  • Hideaki Ando
  • Takayuki Michikawa
  • Katsuhiko MikoshibaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 645)


Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) 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 IP3R isoforms from various cells and tissues using affinity column chromatography and for the specific knockdown of the expression of IP3R 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.

Key words

IP3 Ca2+ Affinity chromatography GST-fusion protein siRNA Ca2+ oscillation IRBIT ERp44 



This work was supported by grants from the Education, Culture, Sports, and Technology of Japan.


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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Akihiro Mizutani
    • 1
  • Katsuhiro Kawaai
    • 1
    • 2
  • Chihiro Hisatsune
    • 1
  • Hideaki Ando
    • 3
  • Takayuki Michikawa
    • 1
    • 4
  • Katsuhiko Mikoshiba
    • 1
    • 4
    Email author
  1. 1.Laboratory for Developmental NeurobiologyBrain Science Institute, RIKENSaitamaJapan
  2. 2.Department of Chemistry and Biological ScienceAoyama-Gakuin UniversityKanagawaJapan
  3. 3.Oregon Stem Cell CenterOregon Health & Sciences UniversityPortlandUSA
  4. 4.Calcium Oscillation Project, International Cooperative Research Project-Solution Oriented Research for Science and TechnologyJapan Science and Technology AgencySaitamaJapan

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