Calcium Functional Assay Services

Creative Biolabs is professional in GPCR ligand-binding assay and provides comprehensive technologies. To help accelerate the progress of the experiment, we will offer appropriate services according to the different needs of customers.

Intracellular Ca²⁺ is another second messenger for GPCR signaling. GPCRs that naturally couple to Gα_q produce a ligand-dependent increase in intracellular Ca²⁺. However, Gα_i/o, Gα_s, or Gα12-coupled GPCRs can also be switched to induce an increase in intracellular Ca²⁺ either by the expression of a chimeric G-protein (Gα_qi5 or Gα_qo5) or a promiscuous G-protein (Gα₁₆ or Gα₁₅).

Fig. 1. Schematic representation of genetically encoded calcium indicator (GECI)-based Ca²⁺ mobilization assay. (Ma, 2017)

Basic Methods of Calcium Functional Assay Services

• Photoprotein-based assays for measurement of intracellular Ca²⁺

The most widely used photoprotein for evaluating intracellular Ca²⁺ signals is aequorin. Aequorin is a Ca²⁺-sensitive photoprotein isolated from the jellyfish Aequorea victoria. It consists of a single 21-kilodalton polypeptide chain containing three potential Ca²⁺-binding sites. In the presence of molecular oxygen from apoaequorin and its cofactor coelenterazine, the binding of Ca²⁺ ions to the three potential Ca²⁺-binding sites causes conformational changes leading to the oxidation of coelenterazine to coelenteramide and the subsequent emission of photons in the blue range of the visible spectrum (λ_max=470nm). Thus, the detection of bioluminescent signals requires no light excitation or any use of fluorescent dyes and does not cause autofluorescence, photobleaching, or any biological degradation problems. In addition to its high sensitivity, undetectable background, and excellent signal-to-noise ratio, aequorin shows no cytotoxicity in most cell types and does not bind significantly to other divalent cations. This makes it a good indicator to sense intracellular Ca²⁺ concentrations with a large dynamic range.

• Measurement of intracellular Ca²⁺ changes using synthetic fluorescent indicators

The most common Ca²⁺ indicators in use today are designed and synthesized based on the structures of the Ca²⁺ chelators, such as EGTA, APTRA, or BAPTA, with the incorporation of a fluorescent moiety. The binding of Ca²⁺ to the Ca²⁺-binding part of the molecule induces the configuration change of the fluorescent moiety, resulting in an acute increase or decrease in fluorescence intensity. In addition, a lipophilic molecule (acetoxymethyl (AM) ester) is conjugated to the charged indicator thus facilitating the transportation into cells. When the Ca²⁺-sensitive indicator-AMs enter the cytosol, the lipophilic groups are cleaved by esterases. This helps retain the indicator within the cell for the detection of Ca²⁺. Before the introduction of synthetic Ca²⁺-sensitive indicators, the main approaches for analyzing intracellular Ca²⁺ concentrations/variations were methods using Ca²⁺-selective microelectrodes and Ca²⁺-activated photoproteins, and they were rather complicated.

Our Feature Service

• Comprehensive technology platform providing one-stop service.
• Professional experts with extensive experience.
• Multiple assay methods depend on a specific project.

With a comprehensive technology platform, Creative Biolabs can meet our customers' different requirements. Our experienced scientists will also help optimize the experiment scheme to ensure to accelerate the objective project. If you are focusing on calcium functional assay, or you have any other questions, please feel free to contact us for more information.

Reference

1. Ma, Q.; et al. An overview of Ca(²⁺) mobilization assays in GPCR drug discovery. Expert Opin Drug Discov. 2017, 12(5): 511-523.