100x TRF-Ubiquitin Mix

Catalog number: SBB-TR0051-2K, 2000 wells

Mixture of Europium Cryptate Ubiquitin (SBB-TR0014), Cy5-Ubiquitin (SBB-TR0015), and wild-type Ubiquitin (SBB-UP0013) combined in a ratio optimized for TR-FRET based conjugation experiments where long polyubiquitin chains are formed. Conjugation reactions are monitored at Excitation = 304 nm, Emission channel 1 = 620 nm, Emission channel 2 = 665 nm. These proteins were expressed in E.coli.

In stock
SKU
0051
$495.00

Description

Ubiquitin is a highly conserved protein that plays a major role in the ubiquitination pathway, which is conserved from yeast to mammals. Ubiquitination, the conjugation of ubiquitin to other proteins through a covalent bond between its C-terminal glycine and the ɜ-amino group of lysine residues (or the ɜ-amino group of an N- terminal methionine) onto proteins is essential for many cellular process primarily linked to protein degradation. This process involves three steps with specific groups of enzymes in an ATP dependent manner, which are activation with ubiquitin-activating enzymes (E1s), conjugation with ubiquitin-conjugating enzymes (E2s), and ligation with ubiquitin ligases (E3s). This product is a mix of Europium Cryptate Ubiquitin (SBB-TR0014), Cy5-Ubiquitin (SBB-TR0015), and wild-type Ubiquitin (SBB-UP0013) in a ratio optimized for TR-FRET based conjugation experiments where long polyubiquitin chains are formed. Under conditions where short Ubiquitin chains are formed, a different mix may be required to optimize signal to background ratio. Enzymatic incorporation of the labeled ubiquitins into chains conjugated onto a substrate protein 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: 2000 wells
Molecular Weight: 8.5 to 8.9 kDa
Purity: >99%
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 Buffer: 50mM Hepes pH 7.5, 100mM NaCl
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 E3 Ligase titration (autoubiquitination): An example using serial dilutions of GST-MDM2 from 200nM to 12.5nM mixed with UBA1, UBE2D3, and, trf-Ub mix. Reaction was initiated with addition of Mg-ATP.

Estimated Z-factor

Figure 2. Estimated Z-primes of Continuous Real-Time TR-FRET E3 Ligase titration (autoubiquitination): Example of serial dilutions of GST-MDM2 from 200nm to 12.5nM mixed with UBA1, UBE2D3, and trf-Ub mix. Reaction was initiated with addition of Mg-ATP.

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).