Fluorescein-Ubiquitin

A single fluorescein moiety is covalently linkedon recombinant human ubiquitin. The N-terminal methionine, the C-terminal glycine, and all lysine residues are available for ubiqiutination. It pairs with Terbium-Ubiquitin (Catalog# T2001) in TR-FRET assays to assess protein ubiquitination.

The TR-FRET assay condition should be optimized, including reaction time and concentrations of UbE1, E2, E3, terbium-ubiquitin and fluorescein-ubiquitin, to achieve a desirable signal-to-background ratio. A typical range of terbium-ubiquitin concentration in TR-FRET assays is 5-30 nM, and fluorescein-ubiquitin concentration is usually 2-15 fold molar excess of terbium-ubiquitin. UbE1, E2 and E3 concentrations are usually at the range of 10-30 nM, 25-250 nM, 10-250 nM, respectively.

100X RELAY^TR Terbium/FluoresceinUbiquitin Mix (Catalog# T2501) with optimized concentration and ratio of these two Ub moieties are used in our RELAY^TR FRET ubiquitination assays.

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

Additional Information

Product Name:

Also Known As:

Fluorescein-Ub; FI-Ub

Catalog No.:

T2201-1000

Size:

1 mg

Molecular Weight:

9 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:

~ 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, MSDS

NCBI RefSeq:

Image(s):

Images:
(Click image to enlarge)

A. Fluorescent visualization of fluorescein-Ub separated on an SDS-PAGE. TR FRET assays with Tb-Ub and Fluorescein-Ub. The ratios (R) of fluorescence intensity at 520 nm and 490 nm from reactions containing E1/E2/E3/Ub Mix/ATP and E1/E2/Ub Mix/ATP were Signal (R_S) and Blank (R_B), respectively. The signal-to-blank ratio was calculated by using the formula of (R_S-R_B)/R_B. Time 0 is ~5 min after ATP addition.
C. Autoubiquitination of 12.5 nM UbE3A detected by RELAY^TR FRET assays.
D. Autoubiquitination of 12.5 nM MURF2 detected by RELAY^TR FRET assays.

Shipping Method:

Dry ice shipping

References:

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

Details

A single fluorescein moiety is covalently linked on recombinant human ubiquitin. The N-terminal methionine, the C-terminal glycine, and all lysine residues are available for ubiqiutination. It pairs with Terbium-Ubiquitin (Catalog# T2001) in TR-FRET assays to assess protein ubiquitination
The TR-FRET assay condition should be optimized, including reaction time and concentrations of UbE1, E2, E3, terbium-ubiquitin and fluorescein-ubiquitin, to achieve a desirable signal-to-background ratio. A typical range of terbium-ubiquitin concentration in TR-FRET assays is 5-30 nM, and fluorescein-ubiquitin concentration is usually 2-15 fold molar excess of terbium-ubiquitin. UbE1, E2 and E3 concentrations are usually at the range of 10-30 nM, 25-250 nM, 10-250 nM, respectively.
100X RELAY^TR Terbium/FluoresceinUbiquitin Mix (Catalog# T2501) with optimized concentration and ratio of these two Ub moieties are used in our RELAYTR FRET ubiquitination assays.
Reaction time is usually 1-3 hours in kinetic or end point assay.