Fluorescein-Ubiquitin

A single fluorescein moiety is covalently labeled 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 10-30 nM, and fluorescein-ubiquitin concentration is usually 6-15 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 were 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.:

T2201-100

Size:

100 µg

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. C. CHIP concentration-dependent autoubiquitination monitored by RELAYTR FRET assays with Tb-Ub and Fluorescein-Ub. S520/S490 ratios from reactions with ATP were positive Signal (S), and without ATP were Backgrounds (B). The signal-to-backbround ratio was calculated using (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 fluorescein moiety is covalently labeled 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 10-30 nM, and fluorescein-ubiquitin concentration is usually 6-15 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 were used in our RELAYTR FRET ubiquitination assays.
Reaction time is usually 1-3 hours in kinetic or end point assay.