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UbiQ news: Ub-rho110MP performance test

By

Wieger Rupert

ubiquitin-rhodamine110MP

performance test

Recently we introduced a new type of quenched ubiquitin rhodamine110 assay reagent, Ub-Rh110MP (UbiQ-126). In this newsletter we present some performance data.


As can be seen in Figures 1-3, Ub-Rh110MP has the:

  • same excellent properties as the classic Ub-Rh110Gly substrate and;
  • increased fluorescence intensity (Figures 1C and 3)

Figure 1: Michaelis-Menten kinetics


UbiQ-126 (Ub-Rh110MP) and UbiQ-002 (Ub-Rh110Gly), turned over by 5 pM UCH-L3. Enzyme kinetics were determined in 384 well format (30 uL per well) on a BMG Clariostar plate reader measuring fluorescence intensity at λexc 487 ± 14 nm; λemi 535 ± 30 nm; 40 flashes per well.  

  1. enzyme velocity represented as AFU/s versus substrate concentration. AFU: arbitrary fluorescence units
  2. enzyme velocity represented as M/s versus substrate concentration 
  3. fluorescence intensities determined at 30 min turnover by 5 pM UCH-L3, error bars are SD (n=3)
 

Figure 2: fluorescence signal versus background


Fluorescence intensities were measured of various concentrations of UbiQ-126 Ub-Rh110MP)(background) and fully converted UbiQ-126 by 1 µM USP7 (signal). 

  1. fluorescence signal of processed UbiQ-126 is linear up to 8 µM. 
  2. signal-to-background ratios over a concentration range of UbiQ-126. 
  3. concentration range of UbiQ-126, determined over 13 replicates. Fluorescence intensities were measured in 384 well format on a BMG Pherastar plate reader at λexc 485 ± 16 nm; λemi 520 ± 10 nm.
 

Figure 3: progress curves of substrate conversion


Substrate conversion of UbiQ-002 (Ub-Rh110Gly) and UbiQ-126 (Ub-Rh110MP) by 1 nM USP7.

  1. conversion of UbiQ-002 (blue dots) at the indicated concentrations.
  2. conversion of UbiQ-126 (red dots) at the indicated concentrations.
Fluorescence intensities were measured in 384 well format on a BMG Pherastar plate reader at λexc  485 ± 16 nm; λ emi 520 ± 10 nm. AFU: arbitrary fluorescence units.
 
 

Ub-Rh110MP (UbiQ-126)

  • increased fluorescence intensity
  • the same excellent properties as the classic Ub-Rh110Gly
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