Recombinant Human UbcH5a/UBE2D1 - Ubiquitin Charged Protein

  
  • Purity
    >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain.
  • Activity

    Pre-charged UBE2D1 may be added directly to in vitro reactions containing E3 Ubiquitin ligases and ligase substrates (if applicable)—no ATP, E1 enzyme, or extra Ubiquitin are required.  Reaction conditions will need to be optimized for each specific application. Note: Reducing agents including dithiothreitol (DTT) or mercaptoethanol ( beta ME) may cause unintended thiolytic release of Ubiquitin from the complex—care must be taken if these compounds are present in buffers. We suggest using a thioester friendly reductant such as TCEP if possible.

  • Source
    E. coli-derived
  • Accession #
  • Predicted Molecular Mass

    25 kDa (17 kDa UBE2D1, 8.6 kDa Ubiquitin)

E2-800
 
Formulation

Approximately 0.43 mg/ml (25 μM) with respect to UBE2D1.  Formulated in 50 mM HEPES pH 7.5, 50 mM NaCl, 1 mM TCEP

Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
Background: UbcH5a/UBE2D1

Ubiquitin-­conjugating Enzyme H5a (UbcH5a), also known as Ubiquitin­-conjugating Enzyme E2D 1 (UBE2D1), is a ubiquitously expressed protein that is related to Stimulator of Iron Transport (SFT).  Human UbcH5a/UBE2D1 has a predicted molecular weight of 17 kDa and shares 89% and 88% amino acid (aa) sequence identity with the related family members UbcH5b and UbcH5c, respectively.  Human UbcH5a/UBE2D1 shares 100% aa sequence identity with the mouse and rat orthologs.  UbcH5a/UBE2D1 has a conserved E2 catalytic core domain that contains an active site cysteine residue, and it interacts with a variety of HECT and RING finger Ubiquitin ligases (E3) to mediate the ubiquitination of c­Fos, RIP1, HIF­1 and other targets.  Pathologically, UbcH5a/UBE2D1 is implicated in protein degradation during cancer and immune responses.

This product is an enzymatically generated UbcH5a/UBE2D1-Ubiquitin thioester complex that has been purified to remove E1 Ubiquitin Activating enzyme, uncharged UbcH5a/UBE2D1, free Ubiquitin, and Mg2+-ATP.  The product provides a convenient starting material for use in single-turnover "Ubiquitin Discharge Assays," eliminating the need to either inhibit the E1 Ubiquitin Activating enzyme with potentially confounding chemical treatments or remove ATP via enzyme additions.

  • References:
    1. Das, R. et al. (2013) EMBO J. 32: 2504
    2. Koyano, F. et al. (2014) Nature 510: 162
    3. Liew, C.W. et al. (2010) Biochem. J. 431: 23
    4. Petroski, M.D. & Deshaies, R.J. (2005) Cell 123: 1107
    5. Saha, A. et al. (2011) Mol. Cell 42: 75
  • Entrez Gene IDs:
    7321 (Human); 216080 (Mouse); 361831 (Rat)
  • Alternate Names:
    E2(17)KB1; Stimulator of Fe transportUBC4/5 homolog; UBC4/5; UBC5A; UbcH5; UbcH5a; UBCH5AEC 6.3.2.19; UBCH5SFT; UBE2D1; Ubiquitin carrier protein D1; ubiquitin-conjugating enzyme E2 D1; Ubiquitin-conjugating enzyme E2(17)KB 1; Ubiquitin-conjugating enzyme E2-17 kDa 1; ubiquitin-conjugating enzyme E2D 1 (UBC4/5 homolog, yeast); Ubiquitin-protein ligase D1
Related Research Areas

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