UbiQ Assay, 탈유비퀴틴 효소(DUB,deubiquitylating enzyme) 활성 측정용 시약

 

 

UbiQ사에서는, 일반적인 amide 결합형과 isopeptide 결합형(fluorescence polarization, FP) 2 종류의 탈유비퀴틴

효소(DUB) 활성 측정용 시약을 공급하고 있습니다

Ubiquitin의 C말단 카르복실기와 리포터 분자의 amino기가 amide 결합을 하게 하여, 탈유비퀴틴 효소(DUB)에

의해서 분해되면 형광을 발합니다.

Ubiquitin (Ub)/Ubiquitin-like proteases(Ubl)과 TAMRA 표지 peptide가, native의 isopeptide bond을 하게 하여,

이 고분자량의 시약은 여기에 의해서 큰 편광을 나타냅니다. 이것은 DUB에 의해 분해되면, 표지 색소는 보다 작은

분자로 분해되어 편광도가 작아지게 됩니다. 본 시약은, DUB 활성을 이러한 편광의 차이에 따라 검출할 수 있습니다.

 

UbiQ-Assay, DUB activity assay reagents, proved effective in multiple industrial scale HTS campaigns. They

are available in various flavors for a wide range of assay read outs, e.g. fluorogenic substrates for

fluorescence intensity read-out, substrates for FP assays, or as highly specialized context specific FP

reagents (our class II FP reagents). All of them targeting DUBs & Ub-like proteases.

 

  

Features

• Substrate supply for an undisclosed number of large library screens by our industrial partners

• Development of >10 highly specialized context specific custom assay reagents

• Demonstrated performance at independent European screening centers – Pivot Park Screening Center and

   European ScreeningPort (table 1)

• Five DUB screening campaigns (>1.7 million small molecules)

• Targets DUBs and Ubl proteases

 

 

Applications

• Compound library screening

• Activity assays

 

 

Table 1. UbiQ’s screening reagents used in HTS campaigns*

Screen#

Primary screen +

dose-response

orthogonal assay

for hit validation

small-molecule

library size

Z'-value

validated hits

hit selected for

dose-response

DUB-001

UbiQ-012 (Ub FP)

UbiQ-002 

(Ub-Rh110)

225K

0.7 - 0.8

293 (0.14%)

99 (0.05%)

310K

0.7 - 0.8

664 (0.21%)

182 (0.06%)

DUB-002

UbiQ-012 (Ub FP)

UbiQ-002 

(Ub-Rh110)

310K

0.7 - 0.8

516 (0.17%)

152 (0.05%)

DUB-003

Ub-like FP

reagent

Ub-like

(AMC)

310K

0.7 - 0.8

1009 (0.33%)

170 (0.05%)

DUB-004

Ub-like FP

Ub-like

(AMC)

310K

0.7 - 0.8

Ongoing

Ongoing

DUB-005

Ub FP class II

reagent

UbiQ-002 

(Ub-Rh110)

310K

0.75 - 0.8

Ongoing

Ongoing

* Completed and ongoing HTS programs in which UbiQ’s FP assay reagents were used for the primary

screen (single point at 10 μM), and dose-response experiments of validated hits. All assays showed

excellent Z' values (>0.7). After confirmation of the hits with the FP substrate, an orthogonal Ub-Rh110Gly

(UbiQ-002) or Ub-like AMC assay was used for validation; the number of hits identified at this stage varied

from 0.33−0.14% of the total library. A subset of compounds (in general 0.05% of the total library) is then

selected for IC50 determination using the FP assay reagent (10 concentration steps from 0.15 nM-5 μM).

 

`

 Amide based DUB activity assay reagents1

 

 Here Ub is linked via an amide bond to a reporter

 part, which becomes fluorescent or enzymatically

 active upon cleavage by a DUB.

 

  Luc, Rh110, AMC read-out

 

 

 

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 Specifications

 

 

 

 AMC:

 Rh110Gly:

 LUC:

 

exc/emi - 380/460nm

exc/emi - 485/535nm

exc/emi - 400/505nm

 

 

 

 

 Isopeptide based fluorescence polarization (FP) DUB activity assay reagents2

 

 Here, Ub/Ubl is linked via a native isopeptide bond to a TAMRA labelled peptide. This high molecular

 weight reagent emits largely polarized light (high mP) when excited. Upon cleavage by a DUB, the dye

 ends up covalently attached to a smaller and faster tumbling (di) peptide, resulting in light that is less

 polarized (low mP). DUB activity is monitored by following this change in polarization.

 

Class I FP

 

Class II FP

 

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  Specifications

 

  Specifications

• native isopeptide bond

• high wavelength read out exc/emi - 550/590 nm

• proven in numerous industrial scale HTS

  campaigns

 

 

 

 

• native isopeptide bond

• high wavelength read out

• exc/emi - 550/590 nm

• proven in numerous industrial scale HTS

  campaigns

• introduces substrate context

• custom design possible

 

 

 

Ordering informations

 

- Regular DUB activity assay reagents

Catalog No.

Description

Size

UbiQ-001

Ub-AMC

100 ug

UbiQ-002

Ub-Rh110Gly

100 ug

UbiQ-036

Ub-aminoluciferin

100 ug

UbiQ-126

Ub-Rh110MP

50 ug