Methods are provided for assaying electrophoretically separated components of sample in a gel. Following an initial separation phase of the sample components into distinct bands within separate zones of the gel, the separated components are held stationary on the gel in a hold mode. During the hold mode, reagent required for an intended assay is applied to the zone of the gel comprising the component to be assayed. The assay is conducted during the hold-mode. A wide range of assays can be conducted on the components in the gel, without the need for a blotting step. Following completion of the assay, the hold mode is removed and, if desired, the sample components are further separated in the gel.