Advantages and disadvantages of different methods for kinetic binding experiments
| Methods | Advantage | Disadvantage | Reference with Respect to GPCRs |
|---|---|---|---|
| Established | |||
| Radioligand binding | Widely applicable | A radioligand with high affinity and selectivity is required | Guo et al., 2014 |
| Good documentation | Possible lack of specificity | ||
| Applicable in membranes, cells, tissue slices | Radioactive waste | ||
| Surface plasmon resonance | Label free with respect to the ligand | Target immobilization required | Aristotelous et al., 2015 |
| Flow through system | Purification and stabilization of protein might be required | Christopher et al., 2013 | |
| Little material required | Often artificial environment | Bocquet et al., 2015 | |
| Real-time detection | |||
| In Development | |||
| Quartz crystal microbalance | Comparable to SPR | Comparable to SPR | Aastrup, 2013 |
| Application in living cells allowing paralleled measurement of target and control cells | Wright et al., 2014 | ||
| Real-time detection | |||
| Fluorescence-based assays for ligand binding | General: Unlike for radioisotope labeling, the addition of fluorescent labels might alter the ligand profile. Therefore, an in-depth pharmacological characterization of the ligand is required! | ||
| Fluorescence intensity | Widely applicable | A fluorescent ligand with high affinity and selectivity is required. | Hill et al., 2014 |
| Applicable in membranes, cells, tissue slices | Possible lack of specificity | ||
| Real-time detection | |||
| Fluorescence anisotropy | No physical separation of bound and free ligand required | Ratiometric assay requires a significant change in the ratio of bound and free ligand. Therefore, a high receptor expression is required. | Veiksina et al., 2014 |
| Technically more demanding than fluorescence intensity measurements | |||
| Fluorescence correlation spectroscopy | Free and bound ligand can be distinguished | Technically demanding, skilled personnel required | Briddon and Hill, 2007; Corriden et al., 2014 |
| Possible to study ligands at single molecule level. Living cells can be used. Real-time detection | Low throughput | ||
| Resonance energy transfer | Different settings are possible | Use of an genetically modified receptor requires pharmacological validation | Castro et al., 2005; Fernandez-Duenas et al., 2012 |
| Binding can be monitored by resonance energy transfer between receptor and ligand by fluorescence or bioluminescence | Each receptor needs to be individually engineered and optimized for this assay | Stoddart et al., 2015 | |
| Indirect binding assay because binding is detected by conformational changes, only agonists can be detected directly | Nikolaev et al., 2006 | ||
| GPCR-based FRET sensors are currently the only settings in which conformational changes during ligand binding can be monitored in real time and living cells | Low throughput | Lohse et al., 2012 |