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From 2010.igem.org

Andreas

IgG protease assay

Me and Elisabeth designed an assay for investigating the IgG protase activity of our BioBrick. In short, the idea behind the assay is as follows:

1. Protein extract is prepared from IPTG-induced cells overexpressing IdeS (IgG protease).
2. α-mouse IgG-peroxidase goat IgG secondary antibodies (Sigma-Aldrich) are bound to mouse IgG-Agarose (Sigma-Aldrich) beads.
3. Excess/unbound secondary antibody are removed by washing with PBS.
4. Protein extract is added and left to incubate ON.
   • This step allows for digestion of IgG-peroxidase, thereby releasing the peroxidase from the agarose beads.
5. After spinning down agarose beads, supernatant is collected.
6. Peroxidase substrate is added to identify presence of released peroxidase in the supernatant.

Procedures
See protocols page.