Determining binding interactions between molecules is the goal of an MST assay and using optimal conditions can help you to get the best results. NanoTemper’s Dr. Beate Kern covers these conditions in this on-demand webinar.
1. Choose a labeling strategy that works best for your target
There are several methods to label your target molecule, and NanoTemper offers a variety of labeling kits spanning different chemistries to covalently label primary amines or cysteine residues, and His-tag specific labeling. For choosing the best method, it’s very helpful to understand the functional groups available on your protein. This could, for example, prevent interfering with an interaction by avoiding the labelling of residues located in the interaction pocket. We recommend that you use the His-Tag Labeling Kit RED-tris-NTA kit whenever possible as it offers purification-free and site-specific labeling of polyhistidine-tagged proteins. Whatever the method you choose though, make sure it matches the fluorescence channel of your Monolith instrument.
2. Whenever possible, label your target with TRIC-sensitive fluorophores
MST is a biophysical technique that measures the strength of the interaction between two molecules by detecting variations in fluorescence signal as a result of an IR-laser induced temperature change. The range of the variation in the fluorescence signal correlates with the binding of a ligand to the fluorescent target.
One major component of the overall MST signal is TRIC (Temperature Related Intensity Change), an effect where the fluorescence intensity of a fluorophore is temperature dependent, and is also influenced by binding events. Some fluorophores show more sensitivity to temperature changes so they are better reporters of binding events when performing MST measurements. To you, this means increased signal-to-noise ratios as well as higher signal amplitudes for most measurements. To exploit this phenomenon, we have created the 2nd Generation labeling kits to maximize TRIC. It’s the best choice as you start your measurements with new targets or if you want to improve the results you see with other RED labeling kits.
3. Ensure optimal buffer conditions
Suboptimal buffers or buffer conditions in MST assays can cause several problems, which may ultimately affect the MST signal. A substandard buffer can decrease signal-to-noise ratio, induce aggregation of your sample and even induce adsorption of target to capillary walls. Some proteins require physiological salt concentrations while others thrive in the absence of salt. Some proteins prefer more acidic and some prefer more basic buffer pH. Do not underestimate the beneficial effect a small amount of detergent such as TWEEN® 20 can have on preventing aggregation of your protein sample. The addition of reducing agents (we recommend glutathione or DTT) can be useful for certain types of proteins. Take-home lesson: buffer optimization is not only useful just for MST assays, but for any biochemical experiment. Using the appropriate buffer conditions makes a great recipe for success.
4. Improve your signal-to-noise ratio
Systematically checking buffer conditions to find one that gives a good signal-to-noise ratio is essential for an MST assay, especially when you are trying a new sample. To do this, try different buffers or buffer conditions and measure each of them three times. You will get an idea of how large your binding amplitude is and how much the signal can vary, even though you are measuring the exact same conditions. There are in principle two strategies to improve the signal to noise ratio. Either by increasing the signal or reducing the noise. Reducing the noise is often an underestimated strategy. Signal variations (noise) are oftentimes the result of molecular instability of your labeled interaction partner. The same way that we as humans have a preference for different room temperatures, every protein has a preference for different buffer compositions. The recommendations given to ensure optimized buffers conditions above can help improve signal-to-noise ratio.
5. Keep target concentration constant and low
To make sure that any observed changes in the fluorescence signal of your sample is due to an interaction and not a concentration difference of your target, its concentration must be kept constant throughout the assay. The optimum target concentration should be in the same range or lower than the expected Kd. In case you don’t know your Kd, just start with a concentration in the lower nM range. Keeping the concentration of one interaction partner well below the Kd is not only an experimental consideration that is important for MST assays. This is a general strategy that is used to simplify Kd determination in biochemical and biophysical assays.
6. Make sure initial fluorescence of all capillaries is the same
Since all capillaries must have the same concentration of target, their initial fluorescence should be the same or approximately the same. When a bigger and random variation appears, it is usually due to an issue of homogeneity of the sample. To deal with this you can:
- Centrifuge samples (10 minutes at > 20,000 xg)
- Add detergent (e.g. 0.05% Tween 20)
- Change buffer or buffer conditions (pH, salt concentration, ionic strength)
Keeping these tips in mind is very useful, but remember having MO. Control software helps too. For every assay, the software will do quality checks, tell you how to interpret your results and give you recommendations for assay optimization. And with version 1.6 you can print the detailed sample preparation instructions and take them to your bench for convenient access.