Parkin E3 Ligase TR-FRET Kit

Catalog number: SBB-KF0036, 400 wells

The kit uses ubiquitin labeled with either Europium-Cryptate or Cyanine5 as FRET pair donor and acceptor fluorophores respectively, completely eliminating the need for antibody based detection setups. Enzymatic incorporation of the labeled ubiquitins into chains conjugated onto Parkin leads to an increase in fluorescence emission at 665 nm (Emacceptor) and decrease at emission wavelength 620 nm (Emdonor).

In stock
SKU
0036
$595.00

Description

South Bay Bio’s Parkin E3 Ligase TR-FRET Kit provides a fast and sensitive method monitoring ubiquitin conjugation onto both wild-type Parkin and a more active mutant W403A in solution, resulting from an enzymatic ubiquitin cascade without the need of running and staining an SDS gel. The kit enables continuous TR-FRET detection of ubiquitin chain formation onto Parkin in a real-time detection setup, or in an end-point configuration if desired. TR-FRET uses the extended fluorescence emission decay lifetimes typical of rare-earth lanthanides to impart a short time-delay between FRET donor excitation and emission. This delay provides a means to separate “true” signal from short-lived background fluorescence, and reduce interference from compound fluorescence and other assay artifacts.
The kit uses ubiquitin labeled with either Europium-Cryptate or Cyanine5 as FRET pair donor and acceptor fluorophores respectively, completely eliminating the need for antibody based detection setups. Enzymatic incorporation of the labeled ubiquitins into chains conjugated onto Parkin leads to an increase in fluorescence emission at 665 nm (Emacceptor) and decrease at emission wavelength 620 nm (Emdonor).

For Research Use Only, Not For Use In Humans.

Specifications

Product Overview
Quantity: 400 wells (20μl / well)
Readout: Endpoint/Kinetic
Label or Dye: Europium Cryptate & Cy5
Detection Method: TR-FRET
Substrate Properties: Protein-Based Substrate. Typical experimental concentration is 1x.
Excitation/Emission (nm): 304/620 & 304/665
Storage Store at −80°C after product arrival. Avoid multiple freeze / thaws. It is recommended to make multiple aliquots after the first thaw.

Figures & Data

% Signal : Background

Figure 1. % Signal to Background of Continuous Real-Time TR-FRET Parkin titration (autoubiquitination): Serial dilutions of Parkin W403A from 50 nM to 3.125 nM and 300 nM wt Parkin were mixed with UBA1, UBE2D3, and trf-Ub (pS65) mix. Reactions were initiated with addition of Mg-ATP.

Estimated Z-factor

Figure 2. Estimated Z-primes of Continuous Real-Time TR-FRET Parkin titration (autoubiquitination): Serial dilutions of Parkin from 50 nM to 3.125 nM mixed with UBA1, UBE2D3, and trf-Ub (pS65) mix. Reaction were initiated with addition of Mg-ATP.s

Citations & References

1) Magennis, S. W., Parsons, S., Pikramenou, Z., Corval, A., & Woollins, J. D. (1999). Imidodiphosphinate ligands as antenna units in luminescent lanthanide complexes.Chemical communications, (1), 61-62.

2) Zheng, N., & Shabek, N. (2017). Ubiquitin Ligases: Structure, Function, and Regulation.Annual Review of Biochemistry, (0).