Biochemical Assay Development for Enzyme Activity, IC50 and Binding Kinetics by PhD Scientists

A biochemical assay puts your compound against the purified target with nothing else in the way: no membrane, no efflux, no serum binding. That isolation is the point. It answers whether the molecule engages the target and how potently, before cellular complexity enters. It is the right tool for enzyme kinetics, binding characterization, selectivity panels and ranking a chemistry series on clean, comparable numbers.

IC50 is the practical potency number under stated conditions. Ki is the binding constant, comparable across labs and substrate concentrations. Mode of inhibition, competitive, noncompetitive or allosteric, tells the chemists what the molecule is doing at the site. We establish Km and Vmax first, because an IC50 measured before the enzyme kinetics are known is a number without a foundation.

Because they measure different worlds. A strong biochemical number with a weak cellular one usually means permeability, efflux or protein binding is taxing the molecule before it reaches the target. The reverse pattern suggests off-target contribution in cells. The disagreement is diagnostic, and reading it correctly saves a chemistry cycle; both assay families run here so the comparison is apples to apples.

Affinity with its parts shown: Kd resolved into the on rate and the off rate, rather than a single endpoint number. Two binders with the same Kd can behave completely differently in vivo if one associates fast and releases fast while the other locks on slowly and stays. Residence time has become a design parameter, and label-free kinetics is how it gets measured, for antibodies, VHH formats and small molecules alike.

Yes, that is the page’s core offer. Where needed, the protein is expressed and purified here, then substrate, buffer system, signal window and controls are developed around your target rather than borrowed from a catalog neighbor. If a catalog kit would honestly serve you better and cheaper, we say that instead.

Yes. 384-well format gives the throughput to run a whole series on one plate set, which is what makes the numbers comparable: same day, same reagent lots, same controls. Chemists get a ranked series they can act on, not a scatter of potencies measured under drifting conditions.

Yes, including against VHH and nanobody formats where conventional bridging struggles. Label-free ADA characterization reports binding kinetics rather than a plate signal, which adds a dimension to immunogenicity work: not just whether ADA bind, but how tightly and how fast they release. It complements the tiered ADA program on the immunogenicity page rather than replacing it.

Around your target’s family. The compound runs against related enzymes or binding partners so potency and selectivity arrive together, with panel membership chosen for the off-target risks your series actually carries. A potency number without its selectivity context is half an answer, and reviewers know it.

Z-prime is the statistical margin between your positive and negative controls, the one number that says whether a screening assay can actually separate hits from noise. A Z-prime above 0.5 is the conventional bar for a screen worth running. We establish it during assay development and report it with every screening dataset, because a ranked list from an assay with poor Z-prime is a list of coin flips.