LifeSensors’ diubiquitin substrates represent a new class of substrates for the continuous fluorescent measurement of true isopeptidase activity. The C-terminus of wild type ubiquitin is conjugated via an isopeptide bond to lysine 63 (K63), 48 (K48), or 11 (K11) of a second ubiquitin molecule with the resultant diubiquitin forming an internally quenched fluorescent FRET pair (IQF). Each ubiquitin is labeled with a single molecule of either a fluorescent reporter (i.e. TAMRA) or a highly efficient quenching fluorophore. Cleavage of the IQF DiUb by deubiquitinases leads to separation of the fluorophore from quencher and subsequent increase in observed fluorescence.
IQF DiUb Variants
There is a remarkable difference between K48- and K63-linked ubiquitin molecules in their three-dimensional structure, which accounts for their individual functions. Beyond differential linkages (e.g. K48, K63, K11), LifeSensors has created subpanels of IQF diubiquitin substrates within each linkage. Because each DUB is likely to recognize and cleave substrates with unique steric considerations, these subpanels vary in location of reporter fluorophore and quencher. It is recommended that each DUB be empirically evaluated against a panel of IQF DiUb substrates to select the the optimal fluorophore/quencher pairing. In order to rapidly determine which of these fluorophore/quencher pairs are optimal for your DUB and/or determined linkage specificity for your DUB, LifeSensors offers a panel of 6 substrates comprised of each K63 and K48-linked substrate available to date. Need bulk quantities? We would be happy to work out a custom price that fits your budget. Please contact us for more information. This product is covered by US patent # 8,518,660. By purchasing this product, the purchaser agrees to comply with the terms of our Limited Use Label License.
Additional Materials Required (see manual for more information):
-384-well (Greiner BioONE 781209) or 96-well assay plates (Greiner BioOne 655076), black
-Fluorescent plate reader
-Filters or monochromators compatible with monitoring the fluorescence of TAMRA (Exc. 540 nm/Emm. 580 nm) are required
-Use of a dichroic mirror with a cutoff in the range of 550-570 is recommended to ensure the optimal signal-to-background
-Control Enzyme (USP2core)
|Variable; but the equivalent of 25 µL of 20 µM
|K48, K63, K11
|50 mM Sodium MES, pH 6.0
|K48-(1-6), K63-(1-6), K11
|IQF DiUbs are stable for 4-6 months at 4ºC. Long term (> 6 months) storage at -80ºC is recommended. Avoid repeated freeze/thaw cycles.