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www.bostonbiochem.com • www.bostonbiochem.eu
Continuous Fluorescent Diubiquitin Substrates:
A New Substrate Discovery Platform
Customizable substrates with true isopeptide bonds between ubiquitin moieties are the
cornerstone of Boston Biochem’s newest continuous assay platform for the study of
deubiquitinating enzymes (DUBs). These fluor-based substrates are superior to current stoptime gel densitometry assays which are not scalable and are time and substrate consuming.
● DUB substrate specificity is influenced by many parameters and variables.
● These include but are not limited to: isopeptide linkages, fluor labels, tags and label/tag positions.
● Due to these variables, there is no universal diubiquitin substrate for all DUBs.
● Variables must be optimized for each DUB.
Contact Boston Biochem to begin the substrate optimization for your DUB
Customizable Diubiquitin Substrates
Over 50 unique diubiquitin building blocks are available to create optimal
substrates for your DUB
Choose from any available:
● Linkage position- Isopeptide bonds available at K48, K63 and K11 positions
● Fluor labels- For FRET, quench pairs,
fluorescent polarization and direct detection
● Affinity tags- For secondary detection
reagent or capture assay formats
● Label/tag positions- Fluor labels and
tags located in multiple, specific positions
allow for >50 distal and anchor ubiquitin
combinations
● Ubiquitin mutations- Incorporation of
lysine and alternative amino acid mutations
Since 2008, Boston Biochem has successfully developed custom diubiquitin substrates and sourced
high throughput screens for the following assay formats:
● Direct or Internal FRET ● DELFIA
● FRET using secondary detection reagents ● ALPHA Screen
● IQP (Internal Quench Pairs) ● Various capture assays
2
Substrate Optimization, A Case Study: USP5 Mediated Hydrolysis of K48-Linked Diubiquitin
Shown below is the optimization process of a K48-linked diubiquitin substrate for USP5. This method is superior to
gel-based, stop-time assays which produce reliable data but are time- and substrate-consuming (Fig. 1). A critical
step in the customization process is determining the optimal label position for each DUB. Varying label positions
results in different efficiencies and signal to background. (Fig. 2).
Gel-based Densitometry
WT diubiquitin K48 linked
Boston Biochem
Boston Biochem
K48-WT: 2.78 s-1
Figure 1. Gel Based Assay of K48linked diubiquitin (UC-200). K48linked diubiquitin (5μM) was incubated with 1 nM USP5 (E-322). Various
time points, as indicated by the data,
were taken and quenched by boiling in SDS-PAGE loading buffer.
After completion of the time course,
all samples are run on SDS-PAGE
and stained with coomassie brilliant
blue protein stain. Once sufficiently
de-stained the gel is scanned into a
densitometry software program and
the pixels for the production of monoubiquitin is determined for each time
point. These pixels are quantitated
by using a mono ubiquitin standard
curve also run on the same gel. Pixels are converted to μM product and
the results are plotted versus time.
There is no universal diubiquitin substrate for all DUBs
Continuous Fluorescence: EDANS/DABCYL K48-linked
Various label positions (POS)
Boston Biochem
Boston Biochem
K48-POS1: 0.076 s-1 , S/B: 5.3
K48-POS2: 0.044 s-1 , S/B: 3.2
K48-POS3: 0.132 s-1 , S/B: 3.4
®
Figure 2: Varying the position
of an internally quenched label
(EDANS/DABCYL) alters the rate
of hydrolysis and signal for each
DUB. USP5 (E-322) catalyzed
hydrolysis of K48-linked diubiquitin
with different sites of labeling. Substrates (250nM) were incubated with
1nM USP5, and hydrolysis was
monitored at ex336nM, em485nM
(EDANS).
Substrate Optimization: Comparison of Different Quench Pairs
The optimal position of EDANS/DABCYL for USP5, as determined in Fig. 2, is K48-POS1. This optimal position
improves signal to background (Fig. 3). Labeling at the same positions with alternative quench pairs (TAMRA/QXL)
further improves efficiency and signal to background (Fig. 4). Optimized quench pairs result in a more efficient substrate, a hydrolysis rate closer to wild-type ubiquitin (Fig. 1) and increased signal to background (Fig. 4).
Continuous Fluorescence:
EDANS/DABCYL K48-POS1
Boston Biochem
Boston Biochem
K48-POS1: 0.076 s-1, S/B: 5.3
Contact Boston Biochem to begin the substrate optimization for your DUB
Continuous Fluorescence:
TAMRA/QXL570 K48-POS1
Boston Biochem
Boston Biochem
K48-POS1: 1.82 s-1 , S/B: 7
®
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Substrate Optimization: Initial Data for K63-Linked Diubiquitin
Further accommodating for DUB specificity, Boston Biochem also customizes our continuous fluor-based substrates
by isopeptide linkage. Shown below is step one in the optimization process for a K63-linked deubiquitin substrate for
USP5 (Fig 5). This process will take the K63-linked substrate through all necessary steps to determine the best labels
and positions to increase rates of hydrolysis and signal to background.
Continuous Fluorescence:
EDANS/DABCYL K63-POS1
Boston Biochem
Figure 5. Hydrolysis of K63-linked
diubiquitin cleaved by USP5 (E-322);
1nM USP5 catalyzed hydrolysis of
400nM K63-linked diubiquitin substrate
(EDANS). The cleavage was monitored
at ex336nM, em485nM.
Boston Biochem
K63-POS1: 0.047 s-1, S/B: 2
Ubiqui-Fluor Substrates: The Current Industry Standard
The Most Trusted Mono-Ubiquitin
Fluorescent DUB Substrates Available
Description:
Mono-Ubiquitin Fluorogenic Substrates
Catalog No.
Size
Ubiquitin-AMC
U-550
50 µg
Ubiquitin-AFC
U-551
50 µg
Ubiquitin Rhodamine110
U-555
50 µg
•
•
•
ll substrates are fully characterized
A
in-house and guaranteed
The most frequently cited source
for DUB substrates
Bulk quantities available with
discounted pricing
Figure 6. Comparison of mono-ubiquitin DUB Substrates.
The indicated concentration of each substrate was incubated
with 2 pM UCHL3 (E-325), and fluorescence was measured
on M5 platereader. Circles and error bars represent the ave.
and std deviation, n = 4. Kinetic constants were calculated by
fitting the Michaelis-Menten equation to plots of steady-state
velocity vs. substrate concentration.
Boston Biochem, Inc.
®
840 Memorial Drive
Cambridge, MA 02139
T 617-576-2210
F 617-492-3565
E [email protected]
www.bostonbiochem.com
www.bostonbiochem.eu
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