Indirect Methods

There are alternative modes of FAC-MS operation that are useful in HTS, secondary screening, and lead development. These modes are designed around monitoring the impact of a ligand (or mixture of ligands) on the breakthrough volume of a pre-selected ligand, which we refer to as an indicator. In a typical experiment, the protein stationary phase is equilibrated with the test ligand by adding it to the running buffer. After an infusion period suitable to ensure equilibrium, the indicator ligand is injected. An accelerated breakthrough for the indicator ligand is observed, to a degree determined by the concentration of the test ligand [10]. The data resulting from these experiments can be linearized according to Eq. (3), where the ligands A1 (with K¿t 1) and A2 (with K¿t2) represent the indicator and test ligand, respectively.

The indexing of 0 to i refers to infusions of ligand A2 at progressively higher concentrations, with the corresponding indicator measurements of V — V0. Appropriate indicator ligands are relatively weak (Kds > 10 mM), allowing for infusion under linear isotherm conditions and rapid indicator kinetics. In this way Eq. (4) applies, and the indicator ligand simply functions as tool to measure the reduction in column capacity due to the test ligand (hence the term "indicator" and not ''competitive'' ligand). Under these conditions, the term within large brackets in Eq. (3) disappears and the most accurate data is generated.

Was this article helpful?

0 0

Post a comment