Recombinant Human UbcH5b/UBE2D2 - Ubiquitin Charged Protein Summary
Pre-charged UBE2D2 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.
25 kDa (17 kDa UBE2D2, 8.6 kDa Ubiquitin)
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins.Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration.The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard.In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
E2-801
| Formulation | Approximately 0.63 mg/ml (25 μM) with respect to UBE2D2. Formulated in 50 mM HEPES pH 7.0, 50 mM NaCl |
| 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.
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Reconstitution Calculator
Background: UbcH5b/UBE2D2
Ubiquitin-conjugating Enzyme H5b (UbcH5b), also known as Ubiquitin-conjugating Enzyme E2D 2 (UBE2D2), is a widely expressed member of the Ubiquitin-conjugating (E2) enzyme family. The protein has a predicted molecular weight of 16.5 kDa. UbcH5b/UBE2D2 localizes to both the nucleus and the cytoplasm. The human protein shares 100% and 92% amino acid sequence identity with the mouse and rat orthologs, respectively. This enzyme has an E2 catalytic core domain that contains an active site cysteine residue that is required for the formation of a thioester bond with Ubiquitin. UbcH5b/UBE2D2 is capable of mediating the formation of Ubiquitin chains linked through Lys11, Lys48, or Lys63. Working with the SCF(Fbxw2) and MDM2/HDM2 Ubiquitin ligases (E3s), UbcH5b/UBE2D2 mediates the ubiquitination and degradation of the transcription factors GCM1 and p53, respectively. Along with UBE2N/Ubc13, UbcH5b/UBE2D2 may have a role in the endocytosis and endolysosomal degradation of MHC class I molecules. Nonproteolytic ubiquitination of TRIM5 alphaby UbcH5b/UBE2D2 has been reported to block HIV reverse transcription. Pathologically, UBE2D family members may be critical targets of cadmium during cadmiuminduced renal toxicity. Additionally, overexpression of UbcH5b/UBE2D2 has been linked to inflammatory bowel disease.
This product is an enzymatically generated UbcH5b/UBE2D2-Ubiquitin thioester complex that has beenpurified to remove E1 Ubiquitin Activating enzyme, uncharged UbcH5b/UBE2D2, 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.
- Das, R. et al. (2013) EMBO J. 32: 2504
- Koyano, F. et al. (2014) Nature 510: 162
- Liew, C.W. et al. (2010) Biochem. J. 431: 23
- Petroski, M.D. & Deshaies, R.J. (2005) Cell 123: 1107
- Saha, A. et al. (2011) Mol. Cell 42: 75
FAQs
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