UBE2A, His-Tag Recombinant

Catalog #

79368

$325 *

Size: 20 µg

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Description

Recombinant full length human UBE2A (ubiquitin-conjugating enzyme E2). This construct contains an N-terminal His-tag. The recombinant protein was affinity purified and is active.

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Synonyms

HHR6A, MRXS30, MRXSN, RAD6A, UBC

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Product Data Gallery

4-20% SDS-PAGE Coomassie Staining

Specific Activity

Product Info

Storage and Usage

Citations

Species

Human

Construct

UBE2A (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, 25% glycerol

17 kDa

Specific Activity

30 pmol/min/µg

Genbank #

NM_003336

UniProt #

P49459

Tag(s)

N-Terminal His-Tag

Background

UBE2A or ubiquitin-conjugating enzyme E2A is a member of the E2 ubiquitin-conjugating enzyme family which is required for post-replicative DNA damage repair and plays an important role in various cellular processes. RAD18 and RAD5 play a central role in mediating physical contacts between the members of the RAD6 pathway. UBE2A is specifically important during chronic low dose ultraviolet exposure to prevent counterproductive DNA checkpoint activation and allow cells to proliferate normally. Loss of the RAD6 error-free PRR pathway results in DNA damage checkpoint-induced G2 arrest in CLUV-exposed cells.

References

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

2. Hishida, T., et al., Nature. 2009; 457: 612-615.

Storage/Stability

At least 6 months at –80°C.

Assay Conditions

UBE2A activity was measured by using the wild type ubiquitin protein diluted in Ubiquitination Buffer [(40 mM Tris pH 7.5), 20 mM MgCl₂, 0.1 mg/ml BSA and 0.5 mM DTT)] to a working concentration of 170 ng/µl (20 µM), in AMP-Glo™ based assay (Promega #V5011). Reaction was initiated by mixing 5 µl of a solution of 2x the final concentrations of UBE2A with 5 µl of a 2x Reaction Cocktail (1 µl of stock solution of ubiquitin, 15 ng/ µl UBA1 + + 50 µM ATP).

The reaction was initiated by mixing 5 µl of 2x UBE2A protein dilutions with 5 µl of 2x Reaction Cocktail for 60 minutes at 37°C. After the plate was equilibrated to Room Temperature (RT), 10 µl of AMP-Glo™ Reagent I was added, mixed for 1-2 minutes and incubated at RT for 60 minutes. AMP-Glo™ Reagent II was diluted 100-fold with Kinase-Glo™ One Solution, and 20 µl were added to each well, mixed for 1-2 minutes and incubated at RT for 30 minutes. The plate was read in a GloMax plate reader (Promega # E7031). The Enzyme Specific Activity was calculated as follows: [ (AMP concentration in µM) * (Reaction Volume in µl)] / Reaction time in min) *(Enzyme amount in mg)] x 10^-3. A blank can be determined from a “no substrate” sample by replacing the enzyme solution 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

79368 Lot 231026

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