In general, there are no limitations for the assay buffer used in Monolith experiments. It is highly recommended to add detergent to all solutions (e.g. 0.005% Tween 20 or 0.1% Pluronic F-127)

Typically, in vitro biochemical 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 most commonly used buffers. Every protein is different and may need specific conditions in order to stay in solution. It can help to try different buffer conditions for Monolith experiments if detection of interactions in a common assay buffer is not optimal. The information given here can serve as a starting point.

Importantly, the buffer composition needs to be constant throughout the serial dilution. Please take care not to introduce buffer gradients when diluting one of the interaction partners.

General buffer composition

Divalent ions like Ca²⁺ and Zn²⁺ cannot be added to PBS 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 particular 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.

Complex bioliquids

Monolith experiments are largely buffer independent and can even be carried out in complex bioliquids such as cell lysate or serum. In these cases, it is important to perform auto-fluorescence tests of the solution in absence of target using the pre-test mode in the MO.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 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.

Buffer composition in LabelFree experiments

In LabelFree experiments, generally avoid buffer components which exert any fluorescence at an emission wavelength of 360 nm. This can be easily tested by comparing a water-filled capillary with a buffer-filled capillary. Please note: this pretest must be performed prior to your binding experiment to ensure that the buffer does not contain any fluorescent substance. Here is a list of some common buffer components which show intrinsic fluorescence in LabelFree experiments:

• Triton-X-100
• Tween 20
• Imidazole
• Proteins with aromatic amino acids (e.g. BSA)
• Some aromatic molecules
• Igepal CA-630 (NP-40)