UBE2E3 (UBCH9), His-Tag Recombinant

Catalog #

79371

$325 *

Size: 20 µg

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Description

Recombinant human UBE2E3 (ubiquitin-conjugating enzyme E2 E3), full length. This construct contains an N-terminal His-tag. This protein was affinity purified.

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Synonyms

UBCH9, UbcM2

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Product Data Gallery

4-20% SDS-PAGE Coomassie Staining

UBE2E3 Activity

Product Info

Storage and Usage

Citations

Species

Human

Construct

UBE2E3 (His-Full Length)

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

25 kDa

Specific Activity

17 pmol/min/µg

Genbank #

NM_006357

UniProt #

Q969T4

Tag(s)

N-terminal His-tag

Background

UBE2E3 also known as UBCH9 or ubiquitin-conjugating enzyme E2E 3 is a member of the E2 ubiquitin-conjugating enzyme family. Ubiquitination, the covalent attachment of the protein ubiquitin (Ub) to other cellular proteins is an important cellular mechanism for targeting abnormal or short-lived proteins for degradation that involves at least three classes of enzymes: ubiquitin-activating enzymes, or E1s, ubiquitin-conjugating enzymes, or E2s, and ubiquitin-protein ligases, or E3s(1). UBE2E3 is highly expressed in skeletal muscle (2). UBE2E3 is essential for the proliferation of RPE-1 cells and is downregulated during RPE layer maturation in the developing mouse eye. These findings indicate that UBE2E3 is a major enzyme in modulating the balance between RPE cell proliferation and differentiation.

References

1. Matuschewski, K., et.al. J. Biol. Chem. 271: 2789-2794, 1996.

2. Ito, K., et.al. Cytogenet. Cell Genet. 84: 99-104, 1999.

Storage/Stability

At least 6 months at -80°C.

Assay Conditions

UBE2E3 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 UBE2E3 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 UBE2E3 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 UBE2E3 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

79371 Lot 240111

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UBE2E3 (UBCH9), His-Tag Recombinant

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