Cy5-Ubiquitin

A single Cy5 moiety is covalently linked 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.

The TR-FRET assay condition should be optimized to achieve a desirable signal-to-blank 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 20-100 nM, and Cy5-ubiquitin concentration is usually 0.2-2X of europium-ubiquitin. UbE1, E2 and E3 concentrations are usually in 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 are used in our E3 autoubiquitination assays.

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

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. In-gel fluorescence visualization of Cy5-Ub in an SDS-PAGE.
B. Autoubiquitination of 25 nM CHIP detected by a RELAY^TR FRET assay with Eu-Ub and Cy5-Ub. The ratios (R) of fluorescence intensities at 655 nm and 620 nm from reactions containing E1/E2/Ub Mix/ATP with and without E3 are Signal (R_S) and Blank (R_B), respectively. The signal-to-blank ratio is calculated by using the formula of (R_S-R_B)/R_B. Time 0 is ~5 min after ATP addition. Data are mean±S.D. of triplicates.
C. Autoubiquitination of 12.5 nM MURF2 detected by a RELAY^TR FRET assay.
D. Autoubiquitination of 50 nM MDM2 detected by a RELAY^TR FRET assay.

Shipping Method:

Dry ice shipping

References:

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

Details

A single Cy5 moiety is covalently linked 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.
The TR-FRET assay condition should be optimized to achieve a desirable signal-to-blank 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 20-100 nM, and Cy5-ubiquitin concentration is usually 0.2-2X of europium-ubiquitin. UbE1, E2 and E3 concentrations are usually in 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 are used in our E3 autoubiquitination assays.
Reaction time is usually 1-3 hours in kinetic or end point assays.