On-bead enzyme assays

A convenient way to analyze an enzyme reaction is an enzymatic assay, whereby either the consumption of a substrate or the making of a product is measured. Enzyme assays may serve to identify an enzyme or to determine the amount or activity of an enzyme. In an on-bead enzyme assay, the enzymatic activity can be determined while the enzyme is immobilized on a bead.

On-bead enzymatic assays

On-bead enzymatic assays are conducted, when the enzyme is immobilized on an affinity resin. For that purpose, the enzyme is frequently genetically fused to a peptide- or protein-tag for immobilization. This way, the enzymatic activity is not compromised by the immunocapture. For example, enzymatic activity can be assessed directly after pull-down from a cell lysate using a GFP-enzyme fusion bound to GFP-Trap®.

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On-bead digestion/ interactome studies

Immunocaptured samples can also be analyzed by mass spectrometry (MS). In contrast to on-bead enzymatic assays, on-bead captured proteins serve as substrates for digestion with a protease that is added to the beads. Following this approach, these proteins are directly amenable to digestion for MS analysis, which is a standard application for interactome studies. In fact, it is advantageous to conduct the trypsination on-bead, because this way it is more likely to retain all possible interaction partners for the MS analysis.

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