On-bead Enzyme 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®.

On-bead digestion/ interactome studies

Samples immunoprecipitated with Nano-Traps can be analyzed by mass spectrometry (MS). 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.

Enzyme activity assay graph

Enzyme activity assay