Tag-Lite, the prefered ligand binding technology
The non-radioactive Tag-lite® solution is quickly becoming the industry standard for studying receptor-ligand binding interactions.
The solution continues to demonstrate its effectiveness in the lab, taking the lead over traditional SPA and radioligand binding assays. Tag-lite’s success in the field is based on benefits it provides that are not found elsewhere:
No separation steps
No radioactive waste
Small quantities of labeled ligands
45 validated receptors, and counting
Cisbio scientists have validated 45 Tag-lite binding assays as of this writing. If the receptor you wish to study is not listed, remember that Cisbio’s team of experienced experts works hand-in-hand with clients to create custom binding solutions. All ligand binding assays listed here include labeled cells and their matching fluorescent ligands.
Tag-lite at the bench: What to expect
Two basic binding protocols – saturation binding and competitive binding – are used to assess equilibrium dissociation constants for the labeled probe (Kd) and the unlabeled compounds (Ki).
Saturation binding assay
To determine Kd, the fluorescent ligand is titrated into a solution containing a fixed amount of labeled cells, and then incubated to equilibrium. The video below provides an overview of the protocol.
Competitive binding assay
To determine Ki, the compound is titrated into a solution containing a fixed concentration of fluorescent ligand and a fixed quantity of cells. At equilibrium, the fraction of labeled ligand bound to the receptor is proportional to the recorded FRET signal. Binding affinities are calculated from this resulting signal. The video below provides an overview of the protocol.
Kinetic binding and residence time
Growing evidence suggests that ligand–receptor binding kinetics is an overlooked factor in drug discovery. Cisbio has developed Tag-lite® kits and services to address the kinetic binding needs of GPCR researchers around the world.
Binding kinetics relates to the rate of association and dissociation of a drug-protein complex. Binding kinetics is thought to be a critical parameter for optimizing the in vivo efficacy of drug candidates. As such, kinetics is receiving increasing attention in drug discovery efforts. Tag-lite combines the flexibility of a radioligand binding with the advantages of homogeneous nonradioactive technologies such as SPR. The homogeneous character of Tag-lite enables binding events to be monitored continually without ever having to stop the reaction.
Motulsky and Mahan described equations that could be used for studying the kinetics of competitive binding. Fitting of data using Tag-lite to these equations allows the determination of the association and dissociation rate constants of the unlabeled competing ligand.
Using Tag-lite and a Motulsky & Mahan equations, the Kon and Koff of 2 muscarinic M1 receptor bindiners were calculated (Atropine and Pirenzepine):
Webinar: The kinetics of drug-receptor binding
Distinguished researchers around the world have already switched from radioligand binding to Tag-lite to handle their kinetic binding needs. One such researcher is Stephen Charlton. Watch this free webinar to hear Stephen discuss the importance of ligand binding in drug discovery.
Determination of association and dissociation rates constants
If, like Stephen Charlton, you are ready to use Tag-lite for all your kinetic needs, this application note is your next step. It describes how binding kinetics studies can be enriched with a Kon, Koff approach by providing critical data on how the association and dissociation rates of a receptor-ligand couple can be assessed with streamlined no-wash Tag-lite assays.
Binding assay: do it yourself
Tag-lite offers freedom and flexibility to build up your assays from target construction to assay development.
A plasmid construction is engineered with Tag-lite plasmids and the gene encoding your protein of interest. Once transfected into cells, these plasmids lead to the expression of a protein fused with the tag. The tagged protein covalently and specifically interacts with Tag-lite substrates.
Build your target with Tag-lite plasmids
Cisbio has optimized a plasmid encoding SNAP bordered by restriction sites, for cloning a gene of interest. By using this plasmid, a construction can be engineered. This construction encodes for the protein of interest and SNAP-Tag in the N or C terminal position.
Label your tagged target with Tag-lite substrates
A selection of substrates labeled with HTRF fluorophores is also proposed for SNAP-tag.
Choose an empty plasmid among the 3 different selection markers: Hygromycin, Neomycin, Zeocin.
Using standard cloning techniques, insert the GPCR gene of interest into the empty plasmid.
Using standard transfection techniques, transiently express the TAG-GPCR of interest in your cell line.
Perform the binding assay you designed and built!