Buffer

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

Importantly, the buffer composition needs to be constant throughout a single TRIC experiment. Ensure that Buffer Gradients are not introduced when diluting one of the interacting partners. It is recommended to test the thermal stability of target proteins in different buffer systems to optimize for target stability in the assay. NanoTemper Technologies offers instrumentation for fast and easy protein quality assessment (Tycho NT.6 on nanotempertech.com).

General buffer composition:

A buffer consisting of physiological pH and salt concentration (e.g. pH 7-8, 150 mM NaCl = 0.9% etc.) is 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. Addition of Detergents may be necessary to help solubilize the protein and prevent it from adsorbing to tubes and/or plastic ware. Starting with a low concentration of Tween®20 (0.005 – 0.05 %) or Pluronic®F-127 (0.1 %) for soluble proteins is recommended, whereas membrane proteins typically require higher detergent concentrations (2-3-fold CMC, e.g. DDM, DM or OG).
PBS cannot be combined with divalent ions like Ca ²⁺ and Zn ²⁺ 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 such as 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 recommended to change buffer pH by +/- 0.3 units if strong aggregation occurs, which cannot be reduced by the addition of detergents.

TRIC analysis in complex bioliquids:

TRIC experiments are largely buffer independent and can be carried out in complex bioliquids such as cell lysate, serum or whole blood. In these cases, it is important to perform auto-fluorescence tests of the solution in absence of target using the experiment template “Single-dose Quick Start” in DI.Control software. 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 buffers (>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:

The vast majority of 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 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 identification of suitable buffer conditions. The NanoTemper Technologies Buffer Exploration Kit (Cat# NT-B001) also contains divalent cations as potential co-factors.