UBE2K (UBC1), His-Tag Recombinant

Catalog #

79373

$325 *

Size: 20 µg

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Description

Recombinant human UBE2K (ubiquitin conjugating enzyme E2 K), full length. This construct contains an N-terminal His-tag. This protein was affinity purified.

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Synonyms

E2-25K, HIP2, HYPG, LIG, UBC1

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Product Data Gallery

4-20% SDS-PAGE Coomassie Staining

UBE2K Activity

Product Info

Storage and Usage

Citations

Species

Human

Construct

UBE2K (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

11 pmol/min/µg

Genbank #

NM_005339

UniProt #

P61086

Tag(s)

N-terminal His-tag

Background

UBE2K also known as UBC1 or ubiquitin-conjugating enzyme E2K belongs to the ubiquitin-conjugating enzyme family which interacts with RING finger proteins. UBE2K can ubiquitinate huntingtin, the gene product for Huntington's disease. Huntingtin is ubiquitinated in peripheral cells, suggesting a role for UBE2K in the degradation of huntingtin (1). UBE2K plays a role in aggregate formation of expanded polyglutamine proteins and the suppression of apoptosis in polyglutamine diseases (2). UBE2K also plays a role in the dislocation of newly synthesized MHC class I heavy chains from the endoplasmic reticulum, and involvement in foam cell formation.

References

1. Kalchman, M. A. et.al. J. Biol. Chem. 271: 19385-19394, 1996.

2. De Pril, R. et al. Mol Cell Neurosci. 2007 Jan;34(1):10-9.

Storage/Stability

At least 6 months at -80°C.

Assay Conditions

UBE2K 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 UBE2K 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 UBE2K 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 UBE2K 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

79373 Lot 240111

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