UBE2O, GST-Tag Recombinant

Catalog #

79374

$325 *

Size: 20 µg

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Description

Recombinant human UBE2O (ubiquitin conjugating enzyme E2 O), full length. This construct contains an N-terminal GST-tag. This protein was affinity purified.

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Synonyms

E2-230K, FLJ12878, KIAA1734

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Product Data Gallery

4-20% SDS-PAGE Coomassie Staining

UBE2O Activity

Product Info

Storage and Usage

Citations

Species

Human

Construct

UBE2O (GST-Full Length)

Host Species/Expression System

Sf9

Purity

≥90%

Format

Aqueous buffer solution

Formulation

50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM Glutathione, 0.1 mM EDTA, 0.25 mM DTT, 0.1 mM PMSF, and 25% glycerol

106 kDa

Specific Activity

25 pmol/min/µg

Genbank #

BC022237

UniProt #

Q9C0C9

Tag(s)

N-terminal GST-Tag

Background

UBE2O or ubiquitin-conjugating enzyme E2O is a part of the ubiquitin-mediated protein degradation pathway in which a thiol-ester linkage forms between a conserved cysteine and the C-terminus of ubiquitin and complexes with ubiquitin. HIV-1 Tat has been identified to have a physical interaction with ubiquitin-conjugating enzyme E2O (UBE2O) in human HEK293 and Jurkat cell lines by using affinity tagging and purification mass spectrometry analyses (1).

References

1. Rumbaugh JA. et.al. Neurobiol Dis, 2012 Aug 25.

Storage/Stability

At least 6 months at -80°C.

Assay Conditions

UBE2O activity was measured by using wild-type ubiquitin diluted in Ubiquitination Buffer to a working concentration of 170 ng/µl, in an AMP-Glo™ based assay (Promega #V5011). The reaction was initiated by mixing increasing amounts of UBE2O with UBA1 (ubiquitin-like modifier activating enzyme 1), BIRC3 (baculoviral IAP-repeat containing protein 3) and ATP in Ubiquitination Buffer (40 mM Tris (pH 7.5), 20 mM MgCl₂, 0.1 mg/ml BSA and 0.5 mM DTT).

First a 2x Master Mix containing 170 ng/ µl of ubiquitin, 15 ng/µl UBA1, 40 ng/µl of BIRC3 and 50 µM ATP was prepared. 5 µl of 2x Master Mix were added to 5 µl of UBE2O serial dilutions prepared at 2x the final desired concentrations in Ubiquitination Buffer, and incubated for 60 minutes at 37°C. The plate was equilibrated to room temperature (RT). 10 µl of AMP-Glo™ Reagent I were added, and the plate was incubated at RT for another 60 minutes. 20 µl of Detection Solution were added to each reaction and the plate was incubated at RT for 30 minutes. The plate was read on a GloMax plate reader (Promega #E7031) using the KinaseGlo Luminescence Protocol. The Enzyme Specific Activity (SA) was calculated as follows: [ [(Concentration of AMP generated in µM) x (Reaction volume in µl)]/ [(Reaction time in min) *(Enzyme amount in mg)] ]* 10^-3. The blank was determined from a “no enzyme” sample by replacing UBE2O with an equal volume of Ubiquitination Buffer.

Instructions for Use

Thaw on ice and gently mix prior to use. DO NOT VORTEX. Perform a quick spin before opening. Aliquot into small volumes and flash freeze for long term storage. Avoid multiple freeze/thaw cycles.

Applications

Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.

Shipping Temperature

-80°C

Warnings

Avoid freeze/thaw cycles

Scientific Category

Ubiquitination

Regulatory Status

Research Use Only

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Datasheets/Protocols

79374 Lot 240111

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UBE2O, GST-Tag Recombinant

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