UBE2D2, His-Tag Recombinant

Catalog #

79370

$325 *

Size: 20 µg

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Description

Recombinant Xenopus UBE2D2 (ubiquitin-conjugating enzyme E2 D2), full length. This construct contains an N-terminal His-tag. This protein was affinity purified.

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Synonyms

UBC4, PUBC1, UBC4/5, UBCH5B, E2(17)KB2

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Product Data Gallery

4-20% SDS-PAGE Coomassie Staining

UBE2D2 Activity

Product Info

Storage and Usage

Citations

Species

Xenopus

Construct

UBE2D2 (His-Full Length) (Xenopus)

Host Species/Expression System

E. coli

Purity

≥90%

Format

Aqueous buffer solution

Formulation

50 mM Sodium Phosphate, pH 7.0, 300 mM NaCl, 150 mM Imidazole, 0.1 mM PMSF, 0.25 mM DTT, and 25% glycerol

17 kDa

Specific Activity

25 pmol/min/µg

Genbank #

NM_001093036

Tag(s)

N-Terminal His-Tag

Background

UBE2D2(UBC4) Protein or ubiquitin-conjugating enzyme E2D2 is a member of the E2 ubiquitin-conjugating enzyme family which functions in the ubiquitination of the tumor-suppressor protein p53, which is induced by an E3 ubiquitin-protein ligase. UBE2D2 specifically ubiquitinates E6AP and in vivo inhibition of UBE2D2 can lead to inhibition of E6-stimulated p53 degradation (1). UBE2D2 could conjugate ubiquitin to target proteins in an E6AP-dependent manner.

References

1. Mondal, S., et al., Anal. Biochem. 2016; 510: 41-51.

2. Rolfe, M., et al., Proc. Nat. Acad. Sci. 1995; 92: 3264-3268.

Storage/Stability

At least 6 months at -80°C.

Assay Conditions

UBE2D2 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 UBE2D2 with UBA1 (ubiquitin-like modifier activating enzyme 1), BIRC7 (baculoviral IAP-repeat containing protein 7) 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, 48 ng/µl of BIRC7 and 50 µM ATP was prepared. 5 µl of 2x Master Mix were added to 5 µl of UBE2D2 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 UBE2D2 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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79370 Lot 240111

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