Recombinant Human Ubiquitin Biotin Protein, CF

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Recombinant Human Ubiquitin Biotin Protein, CF Summary

• Details of Functionality: Biotinylated Recombinant Human Ubiquitin can be conjugated to substrate proteins via the subsequent actions of a Ubiquitin-activating (E1) enzyme, a Ubiquitin-conjugating (E2) enzyme, and a Ubiquitin ligase (E3). Reaction conditions will need to be optimized for each specific application. We recommend using Biotinylated Recombinant Human Ubiquitin in conjunction with native Ubiquitin at a combined concentration of 100-500 μM with a 1:1 to 1:5 ratio of Biotinylated Recombinant Human Ubiquitin:native Ubiquitin. The resulting poly-Ubiquitin chains can be visualized/quantitated with avidin-linked detection reagents.
• Source: E. coli-derived human Ubiquitin protein
• Accession #: P0CG47
• Protein/Peptide Type: Recombinant Proteins
• Gene: UBB
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 8.6 kDa (unlabeled), extent of biotinylation varies by lot.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a solution in deionized water.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain
• Reconstitution Instructions: Reconstitute at 10 mg/mL in an aqueous solution.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Ubiquitin Biotin Protein, CF

• RPS27A
• UBA52
• UBB ubiquitin B
• UBB
• UBC
• Ubiquitin

Background

Ubiquitin is a 76 amino acid (aa) protein that is ubiquitously expressed in all eukaryotic organisms. Ubiquitin is highly conserved with 96% aa sequence identity shared between human and yeast Ubiquitin, and 100% aa sequence identity shared between human and mouse Ubiquitin (1). In mammals, four Ubiquitin genes encode for two Ubiquitin-ribosomal fusion proteins and two poly-Ubiquitin proteins. Cleavage of the Ubiquitin precursors by deubiquitinating enzymes gives rise to identical Ubiquitin monomers each with a predicted molecular weight of 8.6 kDa. Conjugation of Ubiquitin to target proteins involves the formation of an isopeptide bond between the C-terminal glycine residue of Ubiquitin and a lysine residue in the target protein. This process of conjugation, referred to as ubiquitination or ubiquitylation, is a multi-step process that requires three enzymes: a Ubiquitin-activating (E1) enzyme, a Ubiquitin-conjugating (E2) enzyme, and a Ubiquitin ligase (E3). Ubiquitination is classically recognized as a mechanism to target proteins for degradation and as a result, Ubiquitin was originally named ATP-dependent Proteolysis Factor 1 (APF-1) (2,3). In addition to protein degradation, ubiquitination has been shown to mediate a variety of biological processes such as signal transduction, endocytosis, and post-endocytic sorting (4-7).

Ubiquitin modified with Biotin via primary amine coupling results in multiple biotinylated Ubiquitin species modified at the N-terminus, as well as lysine residues. Although having a fully functional C-terminus, lysine modification may limit the ability of this reagent to propagate poly-Ubiquitin chains. Biotinylated Ubiquitin can be detected using avidin-linked reagents.

1. Sharp, P.M. & W.-H. Li. (1987) Trends Ecol. Evol. 2:328.
2. Ciechanover, A. et al. (1980 ) Proc. Natl. Acad. Sci. USA 77:1365.
3. Hershko, A. et al. (1980) Proc. Natl. Acad. Sci. USA 77:1783.
4. Greene, W. et al. (2012) PLoS Pathog. 8:e1002703.
5. Tong, X. et al. (2012) J. Biol. Chem. 287:25280.
6. Wei, W. et al. (2004) Nature 428:194.
7. Wertz, I.E. et al. (2004) Nature 430:694.

Publications for Ubiquitin (U-570)(5)

Publications using U-570

• CW Davies, SE Vidal, L Phu, J Sudhamsu, TB Hinkle, S Chan Rosen, FR Schumacher, YJ Zeng, C Schwerdtfe, AS Peterson, JR Lill, CM Rose, AS Shaw, IE Wertz, DS Kirkpatric, JT Koerber Antibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W Nature Communications, 2021-07-29;12(1):4608. 2021-07-29 [PMID: 34326324] (Bioassay, Human)
• G Fenteany, P Gaur, G Sharma, L Pintér, E Kiss, L Haracska Robust high-throughput assays to assess discrete steps in ubiquitination and related cascades BMC Mol Cell Biol, 2020-03-30;21(1):21. 2020-03-30 [PMID: 32228444] (Bioassay, Human)
• L Zheng, SD Conner PI5P4K? functions in DTX1-mediated Notch signaling Proc. Natl. Acad. Sci. U.S.A., 2018-02-12;0(0):. 2018-02-12 [PMID: 29440432] (Bioassay, Human)
• H Janouskova, G El Tekle, E Bellini, ND Udeshi, A Rinaldi, A Ulbricht, T Bernasocch, G Civenni, M Losa, T Svinkina, CM Bielski, GV Kryukov, L Cascione, S Napoli, RI Enchev, DG Mutch, ME Carney, A Berchuck, BJN Winterhoff, RR Broaddus, P Schraml, H Moch, F Bertoni, CV Catapano, M Peter, SA Carr, LA Garraway, PJ Wild, JP Theurillat Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors Nat. Med., 2017-08-14;23(9):1046-1054. 2017-08-14 [PMID: 28805821] (Bioassay, Human)
• CS Yang, K Jividen, A Spencer, N Dworak, L Ni, LT Oostdyk, M Chatterjee, B Ku?mider, B Reon, M Parlak, V Gorbunova, T Abbas, E Jeffery, NE Sherman, BM Paschal Ubiquitin Modification by the E3 Ligase/ADP-Ribosyltransferase Dtx3L/Parp9 Mol. Cell, 2017-05-18;66(4):503-516.e5. 2017-05-18 [PMID: 28525742] (Bioassay, Human)

Bioinformatics

• Gene Symbol: UBB
• Uniprot:
  • P0CG47()