Ubiquitin (human), (fluorescein labeled)

For use in ubiquitinylation studies.

Lyophilized.

~9.5kDa

≥95% (SDS-PAGE)

Purified by multi-step chromatography.

Reconstitute in minimum volume of DMSO (5-10µL) followed by dilution to required concentration with aqueous buffer of choice (e.g. 1mg/mL in PBS).

Produced in E. coli.

Fluorescein labelled ubiquitin is a useful reagent for studying ubiquitinylation in vitro, where it represents a readily detectable alternative to radio-iodinated ubiquitin. Fluorescein labelled ubiquitin is activated by ubiquitin E1, is a competent substrate for known ubiquitin E2/E3 enzymes (thioester bond formation) and can be conjugated to ubiquitin target proteins (isopeptide bond formation).
 
This product contains a number of multiply fluorescein-labelled ubiquitin species, with [fluorescein]_2-4ubiquitin being the predominant species.

Typical thioester assay set-up: 2.5µM Fluorescein-Ub (BML-UW1240), 100nM Ub-E1 (BML-UW9410), 2.5µM E2 (e.g. His₆-UbcH5a, BML-UW9050), 1U inorganic pyrophosphatase, 5mM Mg-ATP in 20mM TRIS-HCl, pH 7.5, 1mM DTT.  Incubate at 37ºC for 30-60 mins. Analyse by SDS-PAGE followed by fluorescence detection.

P0CG47 (UBB), P0CG48 (UBC), P62979 (RPS27A), P62987 (UBA52)