|structure schematic of human SUMO1 protein made with |
iMol and PDB file 1A5R, an NMR structure; ribbon view
Jakob Suckale at en.wikipedia
The technical note describes how the authors used a bacterial SUMOylation system to create a substrate that had SUMO3 modified with streptavidin conjugated to SUMO 2 which was His-tagged. In order to employ AlphaScreen this intact substrate is mixed with Strep-Tactin donor beads and Nickel-Chelate acceptor beads. Therefore in the absence of protease (or protease activity) the substrate will remain intact and excitation at 680 nm will lead to an emission signal at 520-620 nm; while if protease activity is present a decrease in emission signal will be detected. When run in a 384 well format and detected using the PHERAstar from BMG LABTECH they observed an average Z' = 0.83 indicating that this assay is indeed suitable for HTS applications.
The PHERAstar and CLARIOstar from BMG are both ideally suited to the performance of AlphaScreen as described in this application as either can be equipped with a red diode AlphaScreen laser.
For more information please visit the BMG LABTECH website: www.bmglabtech.com