General Overview

This guide focuses on using a ternary complex formation to detect binding between two unlabeled molecules (molecules B and C in Figure 1 below). Changes to the emission spectrum of a fluorescent reporter molecule A bound to B are used to investigate the interaction of interest, BC.

The primary advantage of this type of ternary assay setup is that neither B, nor C need to be covalently labeled.

This assay works under the assumption that A does not interact with C, and that the complex AB binds equally strong or stronger (lower K_d) than the complex BC.

Figure 1. Ternary interaction of complex AB with ternary ligand C.

Step 1: Measuring the binary interaction (affinity) between the reporter molecule A and B.

The initial step is to investigate whether and how the labeled reporter molecule A binds to molecule B. To achieve this, a constant concentration of A is mixed with a dilution series of B, and a dose-response curve is generated, as illustrated in Figure 2. A final labeled target concentration of 20 nM is a good starting point, but can be reduced if the ternary affinity is expected to be very high. The following instructions assume that the target concentration is not changed from this point on.

The binary affinity information is vital for choosing a proper ratio between labeled reporter molecule A and molecule B for ternary complex formation in Step 2.

When working under the premise of a higher Binary Affinity AB than Ternary Affinity BC (equivalent to Binary K_d (AB) < Ternary K_d (BC)), two main scenarios need to be distinguished:

1. Binary K_d (AB) ≤ target concentration (A):

• Use a 1:2 ratio for A:B in Step 2

2. Binary K_d (AB) > target concentration (A):

• Adjust the binary ligand concentration to 2x K_d for the ternary assay. This ligand concentration roughly leads to 67% of target saturation. Saturation levels can be visually analyzed via the binary dose response curve as seen in Figure 2.

Figure 2. Spectral shift dose response curve of the binary interaction of the fluorescent reporter molecule A with B. K_d(AB) = 25 nM à 20nM labeled target (A) and 50nM binary ligand (B) can be used for the AB complex needed for Step II. This equals roughly 67% target saturation, indicated by the red arrow.

If Binary K_d (AB) > Ternary K_d (BC) this experimental setup cannot be carried out as the concentration of B needed for sufficient saturation will exceed the ternary K_d (BC). A reporter molecule with stronger affinity is required in order to continue. The concept of why target concentration should be lower than the studied K_d is explored in our Target Concentration guide.

For the borderline case of Binary K_d (AB) ≈ Ternary K_d (BC) the A:B ratio can be adjusted to roughly 50% saturation. This way, the concentration of B will be equal to the K_d (BC) still enabling a decent K_d-fit for the ternary binding curve.

If the ternary affinity BC is not known, it is recommended to start with the assumption of Binary K_d (AB) < Ternary K_d and iteratively change the A:B ratio if needed for subsequent experiments.

Step 2: Titrating ternary ligand (C) against binary complex (AB)

In this step the affinity of interest between molecules B and C is measured. For this, the ratio of A:B identified in Step 1 is used at constant concentration and added to a dilution series of C, as depicted in Figure 3. The complex AB does not necessarily have to be pre-formed before the ternary ligand (C) is added. If the binary affinities detected in Step 1 approach the lower nM or even pM range, a 30-minute incubation time should be added, however, in order to allow for equilibration formation.

Figure 3. Ternary interaction of complex AB with ternary ligand C. The graphs below the pictogram indicate molecule concentrations during the assay. A and B are kept constant (with B at slightly higher constant concentration than A) while C is titrated.

Please note that this approach assumes that labeled target A and ternary ligand C do not interact. This premise can be verified by running a binary negative control experiment of C titrated against a constant concentration of labeled target A. A constant spectral shift ratio over all dilution points verifies that A and C do not interact.