IP3/IP1 Functional Assay Services

With years of experience, Creative Biolabs is professional in GPCR ligand-binding assay. We provide both radiometric and non-radiometric technologies to meet customers' different requirements.

Pathway of IP3/IP1 Functional Assay

Stimulation of Gα_q or Gα_i coupled-GPCRs activates phospholipase C (PLC), which hydrolyzes phosphatidylinositol bisphosphate (PIP2) to form two second messengers, inositol 1,4,5-triphosphate (IP3) and DAG. While DAG activates protein kinase C (PKC), IP3 activates the IP3 receptor on the endoplasmic reticulum (ER) resulting in an efflux of Ca²⁺ from the ER to the cytoplasm and an elevation of intracellular Ca²⁺. IP3 is very rapidly hydrolyzed to IP2, then to IP1, and finally to inositol by a series of enzymatic reactions.

Basic Methods of IP3/IP1 Functional Assay

Radiometric Method

The radioactive IP3 assay measures 3 H-inositol incorporations and is a traditional assay for the assessment of PLC activity, but it is not suitable for the screening of large compound collections because it requires a cell wash step and generates radioactive waste. There are a few non-radiometric technologies used for the measurement of IP3, such as the Fluorescence Polarization assay and IP accumulation.

Measuring IP accumulation

Reduction in the energy transfer between acceptor IP1 and a europium-conjugated IP1 antibody as cellular IP1 accumulates and replaces the acceptor IP1 in binding the IP1 antibody. This method utilizes the fact that LiCl inhibits the degradation of IP1, the final step in the inositol phosphate cascade, allowing it to accumulate in the cell and to be measured as a substitute for IP3.

Fluorescence Polarization assay

Fluorescence Polarization assay provides a rapid (1-3 hours) and relatively inexpensive way of measuring ligand binding to IP3R and its fragments in real-time. Fluorescence Polarization assay quantifies the binding of fluorescently labeled inositol 1,4,5-trisphosphate (IP3) to N-terminal fragments of the IP3 receptor. Since it does not require the separation of ligand and protein, it can detect even low-affinity interactions. The assay avoids the use of radioactive materials, it is non-destructive and it can resolve Gibbs free energy (ΔG), enthalpy (ΔH), and entropy (ΔS) changes of ligand binding.

Schematic flowchart for the cAMP assay. Fig. 1. Use of fluorescence polarization to measure ligand binding. (Rossi, 2020)

Our Feature Service

Assays are available using either radiometric or non-radiometric technologies.
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With years of experience accumulated from practice, Creative Biolabs has optimized our GPCR ligand-binding assay services. We are capable of offering both radiometric and non-radiometric technologies to support IP3/IP1 functional assay. If you want any suggestions from our experienced experts or you have any questions about our services, please feel free to contact us for more information.

Reference

Rossi, A. M. and Taylor, C. W. Analyses of Ligand Binding to IP3 Receptors Using Fluorescence Polarization. Methods Mol Biol. 2020, 2091: 107-120.

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