Europium-Ubiquitin

A single europium chelate is covalently labeled on recombinant human ubiquitin (Ub). All lysines, the N-terminal methionine and the C-terminal glycine of Ub are available for ubiquitination. Pairing with Cy5-Ub (Catalog # T3201), this product has been validated in RELAY^TR FRET assays to assess autoubiquitination of E3 Ub ligases, in which excellent signal-to-background ratios were achieved.

The RELAY^TR FRET ubiquitination assay should be optimized to achieve a desirable signal-to-background ratio, including reaction time and concentrations of UbE1, E2, E3, Europium-Ub and Cy5-Ub, A typical range of europium-Ub concentration is 25-100 nM, and Cy5-Ub concentration is usually 0.5-2X of europium-Ub. UbE1, E2 and E3 concentrations are usually at the range of 10-30 nM, 25-250 nM, and 10-250 nM, respectively.

100X RELAY^TR Europium/Cy5 Ubiquitin Mix (Catalog # T3501) with optimized concentration and ratio of these two Ub moieties were used in our E3 autoubiquitination assays.

Reaction time is usually 1-3 hours in kinetic or end point assay.

Additional Information

Product Name:

Europium-Ubiquitin

Also Known As:

Eu-Ub; Eu-Ubiquitin; Europium-Ub

Catalog No.:

T3001-5

Size:

5 µg

Molecular Weight:

9.5 kDa

Species:

Human

Source:

Bacterial recombinant

Stock:

20 mM HEPES, pH 7.2, 50 mM NaCl, 2 mM TCEP

Concentration:

See tube label

Quality Assurance:

~ 95% by SDS-PAGE; Validated by RELAY^TR ubiquitination assays.

Storage:

Store at -80°C; avoid multiple freeze-thaw cycles

PDF Data Sheet:

PDF Datasheet

NCBI RefSeq:

Image(s):

Images:
(Click image to enlarge)

A. Coomassie staind SDS-APAGE with 5 µg Tb-Ub or Eu-Ub.
B. Kinetic mode monitoring 25 nM CHIP autoubiquitination. RELAYTR Europium/Cy5 Ubiquitin Mix (Catalog# T3501) was used in all reactions.
C. CHIP concentration-dependent autoubiquitination monitored by RELAYTR FRET assays with Eu-Ub and Cy5-Ub. S655/S620 ratios from reactions with ATP were positive Signal (S), and without ATP were Backgrounds (B). The signal-to-background ratio was calculated by using the formula of (S-B)/B.
D. Itch concentration-dependent autoubiquitination, similar to C. .

Shipping Method:

Dry ice shipping

References:

1. https://www.bmglabtech.com/en/tr-fret/

Details

A single europium chelate is covalently labeled on recombinant human ubiquitin (Ub). All lysines, the N-terminal methionine and the C-terminal glycine of Ub are available for ubiquitination. Pairing with Cy5-Ub (Catalog # T3201), this product has been validated in RELAY^TR FRET assays to assess autoubiquitination of E3 Ub ligases, in which excellent signal-to-background ratios were achieved.
The RELAY^TR FRET ubiquitination assay should be optimized to achieve a desirable signal-to-background ratio, including reaction time and concentrations of UbE1, E2, E3, Europium-Ub and Cy5-Ub, A typical range of europium-Ub concentration is 25-100 nM, and Cy5-Ub concentration is usually 0.5-2X of europium-Ub. UbE1, E2 and E3 concentrations are usually at the range of 10-30 nM, 25-250 nM, and 10-250 nM, respectively.
100X RELAY^TR Europium/Cy5 Ubiquitin Mix (Catalog # T3501) with optimized concentration and ratio of these two Ub moieties were used in our E3 autoubiquitination assays.
Reaction time is usually 1-3 hours in kinetic or end point assay.