Binding and affinity data are key for characterizing molecular interactions, and with benchtop SPR, it has never been easier. Surface Plasmon Resonance (SPR) is a label-free technology which allows researchers to quantitatively analyze binding between two biomolecules. SPR experiments typically consist of a ligand, the biomolecule that is coupled to the sensor surface, and the analyte, the biomolecule that is in solution and binds to the ligand. The attachment of one of the interactants to the sensor chip surface in the form of a covalent bond or transient by means of capturing is known as immobilization. So how do you choose which molecule should be the ligand and which should be the analyte? Choosing the best ligand for your experiment will help simplify immobilization, maximize signal to noise and minimize non-specific binding. To set up your SPR experiment for the best results, follow this easy guide on how to choose which binding partner should be the ligand and which should be the analyte.
When looking at two different sized biomolecules, using the larger one as the analyte will ensure that the response signal is maximized, since SPR signals are dependent on the mass of analyte bound to the ligand. If a small compound is used as the analyte and a large protein is used as the ligand, relatively large amounts of ligand must be on the sensor chip before a significant signal upon binding of the small compound can be seen.
When carboxyl coupling is being used for immobilization, the purest protein should be used as the ligand to ensure that the surface only contains the target protein of interest. Even if the amount of protein is limited, immobilization uses very small amounts (2-5 μg) of protein per immobilization. The purity of the ligand isn’t as important if the ligand has a tag and you are using a capture immobilization strategy.
3) Number of Binding Sites
Some molecular interactions have more than one binding site. For example, when analyzing antibody interactions, the antibody is a bivalent compound and has two binding sites which can both bind to a target molecule. For a case like this, the antibody should be used as the ligand, otherwise the measured data will not be accurate. If the bivalent molecule is used as the analyte, it will bind to two ligand molecules. The first binding site interaction will give a response and the second binding site interaction will stabilize the complex, but without changing the response. This will result in a decrease in the association rate and a decrease in the dissociation rate compared to the intrinsic affinity of the interaction.
If you are working with a protein that already contains a tag (for example – biotin, his-tag, GST-tag, etc) it is helpful to use the tagged protein as the ligand. The presence of tags makes it easier to immobilize proteins to the surface, and often results in higher activity of the ligand because it is oriented in way that ensures the binding site is accessible. The great news is that Nicoya carries a wide-variety of sensor chips that are compatible with various tags!
5) Non-Specific Binding
It is optimal to use the molecule with the least amount of non-specific binding as the ligand. When working with carboxyl or NTA sensors, it is recommended to use the more negatively charged molecule as the analyte to reduce non-specific binding. Since the carboxyl and NTA sensor chip surfaces are negatively charged, a positively charged analyte will result in more non-specific binding. If you are not sure which molecule will have the least non-specific binding, you can run a quick test to see. If you are having trouble with nonspecific binding, here are 4 ways to reduce non-specific binding.