biochemical assay
A biochemical assay tests a compound against the target protein itself, purified and placed in a tube — stripped of the cell and all its complexity. It is like testing a key on a lock you've taken off the door and laid on the workbench: you see exactly how the key fits the lock, without the surrounding house getting in the way.
Typically a purified target (an enzyme, receptor, or other protein) is combined with its substrate or ligand and the test compound in a defined buffer, and a clean readout reports the result — for an enzyme, the rate of product formation; for a binding interaction, displacement of a labeled ligand. Because the system contains only known components, the data are well-defined, easy to interpret quantitatively, and ideal for measuring exact potency values like IC50 and inhibition constants.
The price of that cleanliness is loss of context. A biochemical assay says nothing about whether the compound can cross a cell membrane, survive metabolism, or hit the target inside a living system, and a purified protein may adopt a conformation or lack partners present in the cell. That is why biochemical assays are usually paired with cell-based assays, which restore biological context at the cost of clarity.
A purified protease is mixed with a fluorogenic substrate; the test compound's IC50 is read directly from how much it slows the fluorescence increase.
With only purified components, a biochemical assay yields clean, quantitative potency.
Potency in a biochemical assay often differs from potency in cells: a compound can be a strong enzyme inhibitor in the tube yet weak in cells if it cannot get inside, or vice versa. The two readouts are complementary, not interchangeable.