AQUApure Phospho-Tetra-Ub Chains (K48, pS65) Protein, CF

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AQUApure Phospho-Tetra-Ub Chains (K48, pS65) Protein, CF Summary

• Modification: p Lys48, p Ser65
• Details of Functionality:

  Ubiquitin chains vary in length, linkage, and function. K48-linked, pS65 Tetra-Ubiquitin chains are ideal for investigating Ubiquitin-­binding proteins and as substrates for Ubiquitin­-specific isopeptidases. Reaction conditions will need to be optimized for each specific application. IMPORTANT: Heating this product in SDS­-PAGE buffer or terminating reactions containing this product with heated SDS­-PAGE buffer could lead to unexpected, high apparent molecular weight banding or smearing on gels that is not representative of product purity. For optimal results, we recommend incubation in SDS­-PAGE buffer + DTT at ≤40°C for 20 minutes prior to gel electrophoresis

• Source: E. coli-derived human Tetra-Ubiquitin protein
• Accession #: P0CG47
• Protein/Peptide Type: AQUApure Recombinant Proteins
• Gene: UBB
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain.

  Ub-AQUA analysis:

  K48: 99.31%

  K11: 0.36%

  K63: 0.19%

  K6 : 0.09%

  All other linkages ≤ 0.03%

  p-S65: 86.1%

Applications/Dilutions

• Dilutions:
  • Bioactivity
• Theoretical MW: 34 kDa.
  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, -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer:

  1 mg/ml (29 μM) in sterile, deionized water

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

  Ub-AQUA analysis:

  K48: 99.31%

  K11: 0.36%

  K63: 0.19%

  K6 : 0.09%

  All other linkages ≤ 0.03%

  p-S65: 86.1%

Notes

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

Alternate Names for AQUApure Phospho-Tetra-Ub Chains (K48, pS65) Protein, CF

• HEL-S-50
• TetraUbiquitin
• Tetra-Ubiquitin
• Ub4
• UBB
• ubiquitin B

Background

Linkage specific phosphorylated Poly-Ubiquitin chains may be used as a substrate for in vitro reactions with deubiquitinating enzymes ("DUB's") that cleave the peptide or isopeptide linkage between adjacent Ubiquitin molecules.  Phosphorylated Poly-Ubiquitin chains can also be used to investigate mechanisms of binding and recognition between the chains and other proteins that contain Ubiquitin-Associated domains (UBAs), Ubiquitin-interacting motifs (UIMs), ZnF's and/or other Ubiquitin-sensing elements.

K48-linked Tetra-Ubiquitin chains phosphorylated at Serine 65 are manufactured using recombinant wild-type human Ubiquitin, linkage-specific enzymes, and PINK1 kinase.  The use of purely enzymatic techniques avoids the potential for contaminating synthetic intermediates. The correctness of linkage and purity of each production lot is assessed using the Absolute Quantitation of Ubiquitin method (Ub-AQUA), an LCMS-based technique that provides extremely accurate information on the composition of Poly-Ubiquitin samples.

1. Heo, J.M., Ordureau A., et al. (2015) Mol. Cell  60: 7
2. Kirkpatrick D.S., et al. (2006) Nat Cell Biol.  8: 700
3. Ordureau, A., et al. (2014) Mol. Cell  56(3): 360
4. Ordureau, A., et al. (2015) Pro. Nat. Acad. of Sci. USA 112: 6637
5. Phu L., et al. (2011) Mol Cell Proteomics  10: M110.003756
6. Wall C.E. et al. (2019) Cell Reports  29: 3280

Publications for Tetra-Ubiquitin (UC-250)(2)

Publications using UC-250

• AK LeBlanc, CN Mazcko, TM Fan, DM Vail, BK Flesner, JN Bryan, S Li, F Wang, S Harris, JD Vargas, JP Govindhara, S Jaganathan, F Tomaino, AK Srivastava, TF Chou, GM Stott, JM Covey, B Mroczkowsk, JH Doroshow Comparative oncology assessment of a novel inhibitor of valosin-containing protein in tumor-bearing dogs Molecular Cancer Therapeutics, 2022-10-07;0(0):. 2022-10-07 [PMID: 35876604] (ELISA Standard, N/A)
• JO Watzlawik, X Hou, D Fricova, C Ramnarine, SK Barodia, TF Gendron, MG Heckman, M DeTure, J Siuda, ZK Wszolek, CR Scherzer, OA Ross, G Bu, DW Dickson, MS Goldberg, FC Fiesel, W Springer Sensitive ELISA-based detection method for the mitophagy marker p-S65-Ub in human cells, autopsy brain, and blood samples Autophagy, 2020-10-28;0(0):1-16. 2020-10-28 [PMID: 33112198] (Western Blot, Human)

Bioinformatics

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