In general, there are no limitations to the assay buffer used in TRIC and Spectral Shift experiments. Usually, in vitro biochemical and biophysical assays are performed at near physiological pH to mimic the native environment of the protein. Phosphate buffered saline (PBS), HEPES, and Tris buffers are the most used buffers. Every protein is different and may need specific conditions to maintain stability in solution. The information provided here can serve as a great starting point for setting up your assay. The ideal method to do buffer optimization, however, is with the Buffer Exploration Kit (Cat# NT-B001) and the Buffer Screen experiment in DI.Control.

Importantly, the buffer composition needs to be constant throughout a single experiment. Please take care not to introduce Buffer Gradients when diluting one of the interaction partners. In general, it is recommended to test the thermal stability of target proteins in different buffers to optimize for target stability in an assay. NanoTemper Technologies provides instrumentation for fast and easy protein quality assessment (Prometheus Panta).

General buffer composition:

A buffer consisting of physiological pH and salt (e.g. pH 7-8, 150 mM NaCl, etc.) conditions is typically recommended as a starting point. While a physiological pH is desired, the required pH level will depend on the interaction partners and their “native” environment. In addition to the appropriate salt and pH level, the addition of Detergents may be necessary to help solubilize the protein and prevent it from sticking to tubes and/or plastic ware. Generally, we recommend starting with a low concentration of Tween®20 (0.005 – 0.05 %) or Pluronic®F-127 (0.1 %) for soluble proteins, whereas membrane proteins typically require higher detergent concentrations (2-3 times CMC, e.g. DDM, DM or OG).

PBS cannot be combined with divalent ions like Ca²⁺ and Zn²⁺ as this will result in precipitation.

HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), a zwitterionic buffering reagent, has negligible binding to Ca²⁺, but it can form radicals under various conditions and should thus be avoided in studies of redox processes in biochemistry.

Tris (2-amino-2-(hydroxymethyl)-propan-1,3-diol) is a primary amine and can therefore form Schiff's bases with aldehydes and ketones; it inhibits various enzymatic reactions (e.g., mitochondrial monoamine oxidase (MAO), alkaline phosphatases, α-amylases, aminopeptidases). It also inactivates diethylpyrocarbonate (DEPC), and it chelates divalent metal ions like Cu²⁺, Ni²⁺, Zn²⁺, and weakly also Ca²⁺ and Mg²⁺.

Buffer pH and protein pI:

At a pH equal to a molecule's isoelectric point (pI) the molecule carries no net electrical charge and is often prone to aggregation. Since it is difficult to determine the exact pI of a folded protein, it is therefore recommended to change buffer pH by +/- 0.3 units if strong aggregation occurs, which cannot be reduced by the addition of detergents.

Analysis of interactions in complex bioliquids:

Spectral Shift and TRIC experiments are largely buffer-independent and can be carried out in complex bioliquids such as cell lysate, serum, or plasma. In these cases, it is important to perform auto-fluorescence tests of the solution in the absence of a target, using the experiment template Single-dose Quick Start in DI.Control. It is also important to consider that not all buffers used for cell lysis are compatible with biomolecule function. For instance, the use of high detergent concentrations or low ionic strength conditions can result in the denaturation of proteins. Moreover, high salt concentrations in lysis buffer (>200 mM), which can be helpful to stabilize the protein of interest, might weaken interactions. It is therefore recommended to use buffers with physiological pH and salt concentrations and low detergent concentrations for interaction studies.

Co-factors:

Most biomolecular interactions require the presence of physiological concentrations of co-factors, such as divalent cations or low molecular weight compounds. While divalent cations such as Mg²⁺ or Ca²⁺ typically aid in the stabilization of the three-dimensional structure of proteins, low molecular weight compounds such as heme or NADPH are often located close to the active site of a biocatalyst mediating enzymatic activity or facilitating ligand binding. Conversely, the absence of co-factors can result in significantly lower affinities or even complete suppression of binding. A careful review of the scientific literature is therefore highly recommended, especially when it comes to the identification of suitable buffer conditions. The NanoTemper Technologies Buffer Exploration Kit (Cat# NT-B001) also contains divalent cations as potential co-factors.