Blog

Binding Kinetics of Glutathione-S-Transferase (GST) Tagged Proteins Using OpenSPR™ GST Sensor Chips

Do you work with GST-tagged proteins? Determining binding affinity and kinetic constants of protein interactions has never been easier. In our application note, we demonstrate how OpenSPR™ GST Sensor Chips can be used to immobilize GST-tagged proteins and determine the binding kinetics between those proteins and their binding partners. Speed up your immobilization process and publish faster with GST Sensor Chips.

Binding of Anti-ubiquitin antibody at concentrations of 7.4 nM, 22.3 nM and 66.7 nM (blue, red and black curves). Solid black lines are the bivalent binding model fits.

Why do we study GST-tagged proteins?

Glutathione S-transferase (GST) is a naturally occurring 26 KDa protein that is found in eukaryotic cells. GSTs conjugate xenobiotics to glutathione (GSH) and therefore have the ability to detoxify a diverse range of environmental toxins including chemotherapeutic agents, drugs pesticides, herbicides, and carcinogens. Many researchers integrate the GST DNA sequence into expression vectors to produce recombinant proteins. The result of expression from this vector is a GST-tagged fusion protein (the functional GST protein is fused to the N-terminus of the recombinant protein). Because GST rapidly folds into a stable and highly soluble protein upon translation, GST-tagged proteins promote greater expression and solubility of recombinant proteins. In addition, GST-tagged proteins can be purified or detected based on the ability of GST to bind its substrate, glutathione (GSH). Since GST-tagged proteins are used in many research techniques including microplate coating, protein interaction pull-down, affinity chromatography and ELISA, Nicoya Lifesciences has created a glutathione functionalized sensor chip that makes immobilization of GST-tagged proteins for surface plasmon resonance analysis quick and easy!

Ligand immobilization of GST-ubiquitin to the GST Sensor Chip

Why do researchers use SPR?

Surface plasmon resonance (SPR) can be used to analyze all types of interactions including protein-protein, protein-small molecule, protein-nucleic acid, protein-aptamer, protein-lipid, carbohydrate-protein (carbohydrate-lectin), carbohydrate-carbohydrate and many more. Techniques such as protein affinity chromatography, sedimentation through gradients, gel filtration columns, sedimentation equilibrium and fluorescence methods have been used to study these interactions. However, many of these techniques require complex labelling steps or expensive, complex instrumentation. Most of these techniques only give endpoint measurements such as yes/no binding or affinity. In recent years, many researchers have turned to surface plasmon resonance (SPR) to characterize their biomolecular interactions as a sensitive and quick approach that also provides real-time measurements. SPR is a label-free technology which allows researchers to quantitatively analyze binding between two biomolecules. SPR technology allows you to determine the kon, koff and KD of interactions, providing deeper insight into binding events compared to other techniques that only give endpoint measurements.

Why is the choice of sensor chip important in the design of an SPR experiment?

To set up your SPR experiment, you must first determine which type of  SPR sensor chip is right for your application. Sensor chips consist of a glass substrate, nanogold coating, and usually a functional chemical coating on the gold surface. The type of sensor chip required depends on your ligand, which needs to be immobilized to the gold surface before you can measure the interaction between your ligand and analyte. Each type of sensor chip has its own advantages and disadvantages depending on whether you choose covalent or capture based coupling, and the surface chemistry used. Fortunately, there are generally inherited benefits to using SPR sensor chips such as reusability which lowers the cost of consumables. The number of times you can reuse the sensor chip with the ligand attached depends on the stability of the ligand. Nicoya Lifesciences has created a glutathione functionalized sensor that makes immobilization of GST-tagged proteins simple. The sensor chips are functionalized with glutathione (GSH), allowing ligands to be directly captured onto the sensor surface via the GST tag with the help of Nicoya Lifescience’s GST Reagent Kit.

Example of using OpenSPR™ GST Sensor Chip to analyze binding kinetics of Glutathione-S-Transferase (GST) tagged proteins

Below is an example of how OpenSPR can be used to immobilize GST-tagged proteins and determine the binding kinetics between those proteins and their binding partners. GST-tagged Ubiquitin was immobilized onto a GST Sensor Chip, and the kinetic binding of Anti-Ubiqutin antibody to Ubiquitin was measured using the OpenSPR™ instrument. A bivalent kinetic interaction model was used to determine the KD to be 95.3 nM. This illustrates the value of surface plasmon resonance analysis in such applications as the strength of the interaction can be quickly and easily quantified with a high level of accuracy. You can find the full study here.

Binding of Anti-ubiquitin antibody at concentrations of 7.4 nM, 22.3 nM and 66.7 nM (blue, red and black curves). Solid black lines are the bivalent binding model fits.

Find out how benchtop SPR can help you publish sooner:

Speak to an application scientist