Recombinant Human Ubiquitin AMC Protein, CF

Catalog # Availability Size / Price Qty
U-550-050
R&D Systems Recombinant Proteins and Enzymes
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Product Details
Citations (46)
FAQs
Reviews (3)

Recombinant Human Ubiquitin AMC Protein, CF Summary

Product Specifications

Activity
Recombinant Human Ubiquitin-AMC is a fluorogenic substrate for some Ubiquitin-specific isopeptidases. Release of AMC fluorescence can be monitored with an excitation wavelength of 345 nm and an emission wavelength of 445 nm. Reaction conditions will need to be optimized for each specific application. We recommend an initial Recombinant Human Ubiquitin-AMC concentration of 0.1-1 μM.
Source
E. coli-derived human Ubiquitin protein
Accession #
Predicted Molecular Mass
8.7 kDa

Product Datasheets

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U-550

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

U-550

Formulation Supplied as a 0.2 μm filtered solution in DMSO.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: Protect from light. Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
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Background: Ubiquitin

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).

This fluorogenic substrate for Ubiquitin hydrolases is based on the C-terminus derivatization of Ubiquitin with 7-amino-4-methylcoumarin (AMC). Ubiquitin-AMC is an exquisitely sensitive substrate for UCHL3 (Km = 0.039 μM) and for Isopeptidase-T (Km = 0.17 - 1.4 μM). Ubiquitin-AMC is useful for studying Ubiquitin hydrolases when detection sensitivity or continuous monitoring of activity is essential.

References
  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.
  8. Dang L.C., et al. (1998) Biochem. 37:1868-1879.
  9. Mason D.E., et al. (2004) Biochem 43:6535-6544.
  10. Stein R.L., et al. (1998) Biochem. 34:12616-12623.  
Entrez Gene IDs
7314 (Human); 298693 (Rat)
Alternate Names
RPS27A; UBA52; UBB ubiquitin B; UBB; UBC; Ubiquitin

Citations for Recombinant Human Ubiquitin AMC Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

46 Citations: Showing 1 - 10
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  1. The BAP1 nuclear deubiquitinase is involved in the nonhomologous end-joining pathway of double-strand DNA repair through interaction with DNA-PK
    Authors: Sato, H;Ito, T;Hayashi, T;Kitano, S;Erdjument-Bromage, H;Bott, MJ;Toyooka, S;Zauderer, M;Ladanyi, M;
    Oncogene
    Species: olive baboon
    Sample Types: Recombinant Protein
    Applications: Bioassay
  2. Targeting BAP1 with small compound inhibitor for colon cancer treatment
    Authors: M Kang, SG Park, SA Lee, S Kim, D Lee, ME Shirbhate, SY Youn, KM Kim, SS Cha, J Kwon
    Scientific Reports, 2023-02-08;13(1):2264.
    Species: Human
    Sample Types: Protein
    Applications: Bioassay
  3. Accelerating inhibitor discovery for deubiquitinating enzymes
    Authors: WC Chan, X Liu, RS Magin, NM Girardi, SB Ficarro, W Hu, MI Tarazona G, CA Starnbach, A Felix, G Adelmant, AC Varca, B Hu, AS Bratt, E DaSilva, NJ Schauer, I Jaen Maiso, EK Dolen, AX Ayala, JA Marto, SJ Buhrlage
    Nature Communications, 2023-02-08;14(1):686.
    Species: Human
    Sample Types: Cell Lysates
    Applications: Bioassay
  4. Targeting USP10 induces degradation of oncogenic ANLN in esophageal squamous cell carcinoma
    Authors: YF Cao, L Xie, BB Tong, MY Chu, WQ Shi, X Li, JZ He, SH Wang, ZY Wu, DX Deng, YQ Zheng, ZM Li, XE Xu, LD Liao, YW Cheng, LY Li, LY Xu, EM Li
    Cell Death and Differentiation, 2022-12-16;0(0):.
    Species: Human
    Sample Types: Recombinant Proteins
    Applications: Bioassay
  5. USP14-regulated allostery of the human proteasome by time-resolved cryo-EM
    Authors: S Zhang, S Zou, D Yin, L Zhao, D Finley, Z Wu, Y Mao
    Nature, 2022-04-27;605(7910):567-574.
    Species: Human
    Sample Types: Recombinant Proteins
    Applications: Bioassay
  6. Repurposing the Pathogen Box compounds for identification of potent anti-malarials against blood stages of Plasmodium falciparum with PfUCHL3 inhibitory activity
    Authors: H Bharti, A Singal, M Saini, PS Cheema, M Raza, S Kundu, A Nag
    Scientific Reports, 2022-01-18;12(1):918.
    Species: Human
    Sample Types: Peptide
    Applications: Bioassay
  7. A molecular sensor determines the ubiquitin substrate specificity of SARS-CoV-2 papain-like protease
    Authors: S Patchett, Z Lv, W Rut, M Békés, M Drag, SK Olsen, TT Huang
    Cell Reports, 2021-09-08;36(13):109754.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Bioassay
  8. Small molecule inhibition of deubiquitinating enzyme JOSD1 as a novel targeted therapy for leukemias with mutant JAK2
    Authors: J Yang, EL Weisberg, X Liu, RS Magin, WC Chan, B Hu, NJ Schauer, S Zhang, I Lamberto, L Doherty, C Meng, M Sattler, L Cabal-Hier, E Winer, R Stone, JA Marto, JD Griffin, SJ Buhrlage
    Leukemia, 2021-07-29;0(0):.
    Species: Human
    Sample Types: Protein
    Applications: Bioassay
  9. Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and FlipGFP-based reporter assay
    Authors: Z Xia, MD Sacco, C Ma, JA Townsend, N Kitamura, Y Hu, M Ba, T Szeto, X Zhang, X Meng, F Zhang, Y Xiang, MT Marty, Y Chen, J Wang
    bioRxiv : the preprint server for biology, 2021-03-16;0(0):.
    Species: Human
    Sample Types: Protein
    Applications: Bioassay
  10. The in-vitro effect of famotidine on sars-cov-2 proteases and virus replication
    Authors: M Loffredo, H Lucero, DY Chen, A O'Connell, S Bergqvist, A Munawar, A Bandara, S De Graef, SD Weeks, F Douam, M Saeed, AH Munawar
    Scientific Reports, 2021-03-08;11(1):5433.
    Species: N/A
    Sample Types: Protease
    Applications: Bioassay
  11. Identification of ebselen and its analogues as potent covalent inhibitors of papain-like protease from SARS-CoV-2
    Authors: E Weglarz-To, JM Tomczak, M Talma, M Burda-Grab, M Giurg, S Brul
    Scientific Reports, 2021-02-11;11(1):3640.
    Species: Human
    Sample Types: Protein
    Applications: Bioassay
  12. Typically inhibiting USP14 promotes autophagy in M1-like macrophages and alleviates CLP-induced sepsis
    Authors: F Xu, Y Ma, W Huang, J Gao, M Guo, J Li, L Kong, G Liang, R Du, Q Xu, X Wu
    Cell Death Dis, 2020-08-20;11(8):666.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Bioassay
  13. Turnip yellow mosaic virus protease binds ubiquitin suboptimally to fine-tune its deubiquitinase activity
    Authors: S Fieulaine, MD Witte, CS Theile, M Ayach, HL Ploegh, I Jupin, S Bressanell
    J. Biol. Chem., 2020-07-30;0(0):.
    Species: Human, Virus
    Sample Types: Recombinant Protein
    Applications: Bioassay
  14. Selective USP7 inhibition elicits cancer cell killing through a p53-dependent mechanism
    Authors: NJ Schauer, X Liu, RS Magin, LM Doherty, WC Chan, SB Ficarro, W Hu, RM Roberts, RE Iacob, B Stolte, AO Giacomelli, S Perera, K McKay, SA Boswell, EL Weisberg, A Ray, D Chauhan, S Dhe-Pagano, KC Anderson, JD Griffin, J Li, WC Hahn, PK Sorger, JR Engen, K Stegmaier, JA Marto, SJ Buhrlage
    Sci Rep, 2020-03-24;10(1):5324.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Bioassay
  15. Nedd8 hydrolysis by UCH proteases in Plasmodium parasites
    Authors: M Karpiyevic, S Adjalley, M Mol, DB Ascher, B Mason, GJ van der He, H Laman, H Ovaa, MCS Lee, K Artavanis-
    PLoS Pathog., 2019-10-28;15(10):e1008086.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  16. Characterizing the PRRSV nsp2 Deubiquitinase Reveals Dispensability of Cis-Activity for Replication and a Link of nsp2 to Inflammation Induction
    Authors: S Zhou, X Ge, C Kong, T Liu, A Liu, P Gao, J Song, L Zhou, X Guo, J Han, H Yang
    Viruses, 2019-09-26;11(10):.
    Species: Virus - PRRSV
    Sample Types: Recombinant Protein
    Applications: Bioassay
  17. Stable Occupancy of the Crimean-Congo Hemorrhagic Fever Virus-Encoded Deubiquitinase Blocks Viral Infection
    Authors: FEM Scholte, BL Hua, JR Spengler, JV Dzimianski, JD Coleman-Mc, SR Welch, LK McMullan, ST Nichol, SD Pegan, CF Spiropoulo, É Bergeron
    MBio, 2019-07-23;10(4):.
    Species: Virus
    Sample Types: Protein
    Applications: Bioassay
  18. Crystal structure and activity-based labeling reveal the mechanisms for linkage-specific substrate recognition by deubiquitinase USP9X
    Authors: P Paudel, Q Zhang, C Leung, HC Greenberg, Y Guo, YH Chern, A Dong, Y Li, M Vedadi, Z Zhuang, Y Tong
    Proc. Natl. Acad. Sci. U.S.A., 2019-03-26;0(0):.
    Applications: Bioassay
  19. The ubiquitin interacting motifs of USP37 act on the proximal Ub of a di-Ub chain to enhance catalytic efficiency
    Authors: N Manczyk, G Veggiani, J Teyra, AW Strilchuk, SS Sidhu, F Sicheri
    Sci Rep, 2019-03-11;9(1):4119.
    Applications: Bioassay
  20. Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15.
    Authors: Teyra J, Singer A, Schmitges F, Jaynes P, Kit Leng Lui S, Polyak M, Fodil N, Krieger J, Tong J, Schwerdtfeger C, Brasher B, Ceccarelli D, Moffat J, Sicheri F, Moran M, Gros P, Eichhorn P, Lenter M, Boehmelt G, Sidhu S
    Structure, 2019-01-31;0(0):.
    Species: Human
    Sample Types: Transfected Whole Cells
    Applications: Bioassay
  21. Ubiquitination of UVRAG by SMURF1 promotes autophagosome maturation and inhibits hepatocellular carcinoma growth
    Authors: X Feng, Y Jia, Y Zhang, F Ma, Y Zhu, X Hong, Q Zhou, R He, H Zhang, J Jin, D Piao, H Huang, Q Li, X Qiu, Z Zhang
    Autophagy, 2019-01-27;0(0):1-20.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Bioassay
  22. A new gold(I) complex-Au(PPh3)PT is a deubiquitinase inhibitor and inhibits tumor growth
    Authors: X Li, Q Huang, H Long, P Zhang, H Su, J Liu
    EBioMedicine, 2018-12-05;0(0):.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Bioassay
  23. The novel anti-androgen candidate galeterone targets deubiquitinating enzymes, USP12 and USP46, to control prostate cancer growth and survival
    Authors: UL McClurg, M Azizyan, DT Dransfield, N Namdev, NCTH Chit, S Nakjang, CN Robson
    Oncotarget, 2018-05-18;9(38):24992-25007.
    Applications: Bioassay
  24. USP15-dependent lysosomal pathway controls p53-R175H turnover in ovarian cancer cells
    Authors: A Padmanabha, N Candelaria, KK Wong, BC Nikolai, DM Lonard, BW O'Malley, JS Richards
    Nat Commun, 2018-03-28;9(1):1270.
    Species: Human
    Sample Types: Protein
    Applications: Bioassay
  25. TRIM11 activates the proteasome and promotes overall protein degradation by regulating USP14
    Authors: L Chen, G Zhu, EM Johns, X Yang
    Nat Commun, 2018-03-26;9(1):1223.
    Species: Human
    Sample Types: Protein
    Applications: Bioassay
  26. Reactive-site-centric chemoproteomics identifies a distinct class of deubiquitinase enzymes
    Authors: DS Hewings, J Heideker, TP Ma, AP AhYoung, F El Oualid, A Amore, GT Costakes, D Kirchhofer, B Brasher, T Pillow, N Popovych, T Maurer, C Schwerdtfe, WF Forrest, K Yu, J Flygare, M Bogyo, IE Wertz
    Nat Commun, 2018-03-21;9(1):1162.
    Species: Human
    Sample Types: Protein
    Applications: Bioassay
  27. Ubiquitin C-Terminal Hydrolase L1 regulates autophagy by inhibiting autophagosome formation through its deubiquitinating enzyme activity
    Authors: C Yan, H Huo, C Yang, T Zhang, Y Chu, Y Liu
    Biochem. Biophys. Res. Commun., 2018-02-17;0(0):.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Bioassay
  28. A mobile loop near the active site acts as a switch between the dual activities of a viral protease/deubiquitinase
    Authors: I Jupin, M Ayach, L Jomat, S Fieulaine, S Bressanell
    PLoS Pathog., 2017-11-08;13(11):e1006714.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Bioassay
  29. Characterization of the deubiquitination activity and substrate specificity of the chicken ubiquitin-specific protease 1/USP associated factor 1 complex
    Authors: H Zheng, M Wang, C Zhao, S Wu, P Yu, Y Lü, T Wang, Y Ai
    PLoS ONE, 2017-11-01;12(11):e0186535.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Bioassay
  30. Ubiquitin Recognition of BAP1: Understanding its Enzymatic Function
    Authors: P Hanpude, S Bhattachar, AK Singh, TK Maiti
    Biosci. Rep., 2017-10-27;0(0):.
    Applications: Enzyme Assay
  31. An inhibitor of the proteasomal deubiquitinating enzyme USP14 induces tau elimination in cultured neurons
    Authors: M Boselli, BH Lee, J Robert, MA Prado, SW Min, C Cheng, MC Silva, C Seong, S Elsasser, KM Hatle, TC Gahman, SP Gygi, SJ Haggarty, L Gan, RW King, D Finley
    J. Biol. Chem., 2017-09-26;0(0):.
    Species: Human
    Sample Types: Protein
    Applications: Enzyme Assay
  32. Bilirubin neurotoxicity is associated with proteasome inhibition
    Authors: H Huang, M Guo, N Liu, C Zhao, H Chen, X Wang, S Liao, P Zhou, Y Liao, X Chen, X Lan, J Chen, D Xu, X Li, X Shi, L Yu, Y Nie, X Wang, CE Zhang, J Liu
    Cell Death Dis, 2017-06-15;8(6):e2877.
    Applications: Enzyme Assay
  33. Generation and Validation of Intracellular Ubiquitin Variant Inhibitors for USP7 and USP10
    Authors: W Zhang, MA Sartori, T Makhnevych, KE Federowicz, X Dong, L Liu, S Nim, A Dong, J Yang, Y Li, D Haddad, A Ernst, D Heerding, Y Tong, J Moffat, SS Sidhu
    J. Mol. Biol., 2017-06-03;0(0):.
    Applications: Bioassay
  34. Chlamydia trachomatis-containing vacuole serves as deubiquitination platform to stabilize Mcl-1 and to interfere with host defense
    Authors: A Fischer, KS Harrison, Y Ramirez, D Auer, SR Chowdhury, BK Prusty, F Sauer, Z Dimond, C Kisker, P Scott Heft, T Rudel
    Elife, 2017-03-28;6(0):.
    Applications: Enzyme Assay
  35. A Wolbachia deubiquitylating enzyme induces cytoplasmic incompatibility
    Authors: JF Beckmann, JA Ronau, M Hochstrass
    Nat Microbiol, 2017-03-01;2(0):17007.
    Applications: Bioassay
  36. Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila
    Authors: Malene L Urbanus
    Mol. Syst. Biol, 2016-12-16;12(12):893.
    Applications: Bioassay
  37. p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1
    Proc Natl Acad Sci USA, 2016-08-12;113(35):E5192-201.
    Species: Human
    Sample Types: Protein
    Applications: Enzyme Assay
  38. Saturation scanning of ubiquitin variants reveals a common hot spot for binding to USP2 and USP21
    Proc Natl Acad Sci USA, 2016-07-19;113(31):8705-10.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Enzyme Assay
  39. BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation
    Proc Natl Acad Sci USA, 2016-06-20;113(27):7655-60.
    Applications: Bioassay
  40. Deubiquitination and Activation of AMPK by USP10
    Authors: M Deng, X Yang, B Qin, T Liu, H Zhang, W Guo, SB Lee, JJ Kim, J Yuan, H Pei, L Wang, Z Lou
    Mol. Cell, 2016-02-11;61(4):614-24.
    Applications: Enzyme Assay
  41. X-ray Structural and Functional Studies of the Three Tandemly Linked Domains of Non-structural Protein 3 (nsp3) from Murine Hepatitis Virus Reveal Conserved Functions.
    Authors: Chen Y, Savinov S, Mielech A, Cao T, Baker S, Mesecar A
    J Biol Chem, 2015-08-19;290(42):25293-306.
    Applications: Enzyme Assay
  42. The human otubain2-ubiquitin structure provides insights into the cleavage specificity of poly-ubiquitin-linkages.
    Authors: Altun M, Walter T, Kramer H, Herr P, Iphofer A, Bostrom J, David Y, Komsany A, Ternette N, Navon A, Stuart D, Ren J, Kessler B
    PLoS ONE, 2015-01-15;10(1):e0115344.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Bioassay
  43. Small-molecule RA-9 inhibits proteasome-associated DUBs and ovarian cancer in vitro and in vivo via exacerbating unfolded protein responses.
    Authors: Coughlin K, Anchoori R, Iizuka Y, Meints J, MacNeill L, Vogel R, Orlowski R, Lee M, Roden R, Bazzaro M
    Clin Cancer Res, 2014-04-11;20(12):3174-86.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Bioassay
  44. The vOTU domain of highly-pathogenic porcine reproductive and respiratory syndrome virus displays a differential substrate preference.
    Authors: Deaton M, Spear A, Faaberg K, Pegan S
    Virology, 2014-03-15;454(0):247-53.
  45. Dopamine D3 receptor inhibits the ubiquitin-specific peptidase 48 to promote NHE3 degradation.
    Authors: Armando I, Villar V, Jones J, Lee H, Wang X, Asico L, Yu P, Yang J, Escano C, Pascua-Crusan A, Felder R, Jose P
    FASEB J, 2013-12-05;28(3):1422-34.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  46. A role for intersubunit interactions in maintaining SAGA deubiquitinating module structure and activity.
    Authors: Samara N, Ringel A, Wolberger C
    Structure, 2012-07-05;20(8):1414-24.
    Applications: Bioassay

FAQs

  1. Is Recombinant Human ISG15 Fluorescein Protein, CF (Catalog # UL-613) a suitable substrate for Recombinant His6-SARS Virus Papain-like Protease Protein, CF (Catalog # E-610)?

    • Recombinant His6-SARS Virus Papain-like Protease Protein, CF (Catalog # E-610) only cleaves at the C-terminus of ISG15 and/or Ubiquitin conjugates. Recombinant Human ISG15 Fluorescein Protein, CF (Catalog # UL-613) is not a suitable substrate because the fluorescein-labeled lysine residues are distributed throughout the protein, but not at the C-terminus. Suitable substrate candidates for catalog # E-610 include include Recombinant Human Ubiquitin AMC Protein, CF (Catalog #U-550), Recombinant Human Ubiquitin Rhodamine 110 Protein, CF (Catalog # U-555), and Recombinant Human ISG15 AMC Protein, CF (Catalog # UL-553).

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Reviews for Recombinant Human Ubiquitin AMC Protein, CF

Average Rating: 4 (Based on 3 Reviews)

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Recombinant Human Ubiquitin AMC Protein, CF
By Anonymous on 04/23/2021
Application: Enzymatic activity in vitro
Reason for Rating: A good pseudosubstrate for many DUBs

Recombinant Human Ubiquitin AMC Protein, CF
By Anonymous on 04/22/2021
Application: Enzymatic activity in vitro
Reason for Rating: Works well but is expensive when working with DUBs with low activity in vitro.

Hydrolysis by USP8


Recombinant Human Ubiquitin AMC Protein, CF
By Tao An on 06/01/2019
Application: Enzymatic activity in vitro
Reason for Rating: The substrates was able to be hydrolysed by USP7 and fluoresence was detected.