Cy5-Ubiquitin

A single Cy5 moiety is covalently labeled on recombinant human ubiquitin (Ub). All lysines, the N-terminal methionine and the C-terminal glycine of ubiquitin are available for ubiquitination. It pairs with Europium-Ubiquitin (Catalog# T3001) in TR-FRET assays assessing formation of polyubiquitin cbains (see Results section above).

The TR-FRET assay condition 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-ubiquitin concentration in TR-FRET assays is 25-100 nM, and Cy5-ubiquitin concentration is usually 0.5-2X of europium-ubiquitin. 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:

Cy5-Ubiquitin

Also Known As:

Cy5-Ub; Cyanine 5 ubiquitin

Catalog No.:

T3201-100

Size:

100 µg

Molecular Weight:

9.1 kDa

Species:

Human

Source:

Bacterial recombinant

Stock:

20 mM Tris, pH 7.6 at 4 °C, 150 mM NaCl, and 10% glycerol.

Concentration:

See tube label

Quality Assurance:

>90% by SDS-PAGE; Validated by RELAY^TR ubiquitination assays.

Storage:

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

PDF Data Sheet:

PDF Datasheet, MSDS

NCBI RefSeq:

Image(s):

Images:
(Click image to enlarge)

A. Fluorescent visualization of Cy5-Ub separated on an SDS-PAGE.
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. Nedd4 concentration-dependent autoubiquitination, similar to C.

Shipping Method:

Dry ice shipping

References:

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

Details

A single Cy5 moiety is covalently labeled on recombinant human ubiquitin (Ub). All lysines, the N-terminal methionine and the C-terminal glycine of ubiquitin are available for ubiquitination. It pairs with Europium-Ubiquitin (Catalog# T3001) in TR-FRET assays assessing formation of polyubiquitin cbains (see Results section above).
The TR-FRET assay condition 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-ubiquitin concentration in TR-FRET assays is 25-100 nM, and Cy5-ubiquitin concentration is usually 0.5-2X of europium-ubiquitin. 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.