Membrane Protein Assays
Ligand Binding Assays

CALIXAR offers the access to cutting-edge technologies and  to characterize ligand interaction with any membrane protein targets. Our ligand binding assays services help scientists and researchers who want to obtain accurate ligand binding parameters, compare several ligands in term of binding properties or just identify a specific interaction of their ligands with the membrane target of interest.

MicroScale Thermophoresis with Isothermal Spectral Shift

With the first Monolith X (Nanotemper) installed in France, CALIXAR increases its analytical capabilities in ligand binding to any type of membrane protein. Scientists and researchers are assured of having access to the latest technologies to characterize membrane targets.

Thanks to the MicroScale Thermophoresis (MST) combined with isothermal spectral shift detection, CALIXAR offers to determine in solution interactions of membrane proteins with unlabeled ligands. Binding measurements can be performed using purified membrane targets or proteins in complex environment such as cell membrane.

Monolith X
Detection :
  • Red fluorescence
  • Spectral Shift
  • Dynamic range: 1nm to mM
    Detected molecule range: 101 – 107 Daltons
    Samples per run: Up to 24

    A2AR ligand binding assays using microscale thermophoresis (MST) combined with isothermal spectral shift.

    Ligand binding assays were performed on full-length wild-type A2A adenosine receptor either in cell membrane fractions (A) or purified in solution (B) and with CGS21680 agonist or ZM241385 inverse agonist using a Monolith X (Nanotemper) combining microscale thermophoresis with isothermal spectral shift detection.

    A) A2A adenosine receptor in cell membrane

    ZM241385 inverse agonist (Kd 25nM)

    B) Purified full-length wild-type A2A adenosine receptor

    CGS21680 agonist (Kd 200nM)
    ZM241385 inverse agonist (Kd 2nM)

    Radioligand Binding Assay

    CALIXAR offers ligand binding characterization to any membrane protein using radiolabeled [3H]-ligand.

    The well-established and robust radioactive method enables high-sensitive detection of membrane protein / ligand interactions. Binding measurements can be performed using purified membrane targets or proteins in complex environment such as cell membrane.

    Filtermat-96 Cell Harvester with MicroBeta2 Microplate Counters.

    Simple simultaneous analysis of samples from 96- well microplates for filtration-based receptor binding assays. Radiometric 3H-labeled ligands detection is ensured with high efficiency and extremely low background.

    MicroBeta2 Microplate Counters
    Filtermat-96 Cell Harvester

    A2AR radioligand binding assays.

    Saturation radioligand binding assays were performed on purified full-length wild-type A2A adenosine receptor with [3H]-CGS21680 agonist (A) or [3H]-ZM241385 inverse agonist (B).

    A) CGS21680 agonist (Kd 200nM)

    B) ZM241385 inverse agonist (Kd 4nM)

    Fluorescence Polarization

    CALIXAR identifies challenging protein-ligand interactions by using Fluorescence Polarization.

    Fluorescence polarization is a method to measure protein-ligand interactions by detection the changes of small molecule rotation. When a fluorescently labeled small ligand is bound to a bigger protein the emitted light is polarized due to the slow movement of the complex .

    Purified Multidrug Efflux Pump subunit AcrB

    Ligand binding assay was performed on purified full-length wild-type AcrB with Rhodamine G6 ligand using fluorescence polarization.

    Activity measured by binding assay (fluorescence polarization) Binding of Rhodamine G was measured on purified AcrB. A KD of 6µM was determined for Rhodamine G6.

    AcrB: Rhodamin G6

    Kd: 6 µM

    Purified Voltage-Gated Sodium Channel (Nav1.7)

    Ligand binding assay was performed on purified full-length wild-type Nav1.7 ion channel with protoxin-II (Cy5-ProTx-II) using fluorescence polarization.

    The polarization values of 400nM Cy5-ProTx-II (Smartox) were measured after incubation for 90min at RT in presence of a range of concentrations from 0 to 200nM of native Nav1.7 (in blue) or denatured Nav1.7 (in orange). Nav1.7 was denatured by heating the purified protein at 98°C for 15min. Lower concentrations of protein and ligand could not be explored due to the limited sensitivity of the measurement, preventing the determination of kinetic constants such as the Kd.
    Positive controls in blue (10 replicates) contain 400nM Cy5-ProxTx-II with 200nM Nav1.7 in PBS buffer. Negative controls in orange (10 replicates) were 400nM Cy5-ProxTx-II in PBS buffer. The Z’-factor was determined using the polarization values from 10 repeats of negative and positive controls using the method of Zhang et al.1. The Z’-factor for the measurement was 0.82 and is an indication of a good performance for the assay.

    Get access to CALIXAR's Membrane Protein Assays Technology

    All of our clients receive access to our proprietary technology and know-how (8 patent families, more than 28 publications).

    The CALIXAR® platform uses patented technology only possible for clients under explicit agreements (license, co-development, and services).

    You can leverage CALIXAR’s proprietary technology as well as other leading-in-class membrane protein technology that is not found anyplace else on the market.

    Gain access to our specific expertise with a strong track record and several types of membrane proteins. We are continuously integrating best-of-the-art membrane protein characterization technology for our clients’ projects. Our clients have access to all assays needed for their projects, all on one platform.

    • Save time and money by using our unique assay services all in the same place.
    • Possibility to develop customized assays (transferable to our clients).
    • Possibility to validate customers’ hits/leads and the quality of the proteins produced.