Real-time bio-layer interferometry ubiquitination assays as alternatives to western blotting.
Auto-ubiquitination
Bio-layer interferometry
Western blotting
Journal
Analytical biochemistry
ISSN: 1096-0309
Titre abrégé: Anal Biochem
Pays: United States
ID NLM: 0370535
Informations de publication
Date de publication:
15 10 2023
15 10 2023
Historique:
received:
30
05
2023
revised:
13
08
2023
accepted:
18
08
2023
pmc-release:
15
10
2024
medline:
8
9
2023
pubmed:
22
8
2023
entrez:
21
8
2023
Statut:
ppublish
Résumé
Ubiquitination is a crucial cellular pathway enabling normal cellular functions. Abnormalities in the ubiquitination process can lead to cellular dysfunction and cause a range of diseases. Efforts to screen and develop small molecule inhibitors targeting portions of the ubiquitination cascade require rapid and robust methods for detecting ubiquitination. Enormous efforts have been made in the field to detect ubiquitination using various techniques including fluorescence, spectrophotometry, chemiluminescence, NMR, and radioactive tracers. The most common method to detect ubiquitination is western blotting. However, western blotting is time-consuming and difficult to use when seeking fine-grained time course experiments. Here we present the use of bio-layer interferometry to rapidly assay ubiquitination in real-time. An E3 ligase auto-ubiquitination system and a substrate ubiquitination assay have been applied as tests for the newly developed assay. The developed BLI ubiquitination assay provides one-second time resolution and detects the formation of polyubiquitin chains directly on a biosensor-bound target. Results are returned instantaneously, and reagent concentrations are identical to those used by traditional western blot-based ubiquitination assays. The developed BLI ubiquitination assay is a viable alternative to traditional western blot assays to detect ubiquitination in a rapid real-time manner.
Identifiants
pubmed: 37604387
pii: S0003-2697(23)00261-0
doi: 10.1016/j.ab.2023.115296
pmc: PMC10529061
mid: NIHMS1926511
pii:
doi:
Substances chimiques
Ubiquitin-Protein Ligases
EC 2.3.2.27
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
115296Subventions
Organisme : NIGMS NIH HHS
ID : R35 GM128595
Pays : United States
Informations de copyright
Copyright © 2023 Elsevier Inc. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Richard C. Page reports financial support was provided by the National Institutes of Health.
Références
Cell. 1996 Mar 22;84(6):813-5
pubmed: 8601303
Anal Bioanal Chem. 2021 Feb;413(4):1159-1171
pubmed: 33236226
Front Chem. 2020 Jan 10;7:915
pubmed: 31998699
Molecules. 2020 Nov 09;25(21):
pubmed: 33182242
STAR Protoc. 2021 Sep 15;2(4):100836
pubmed: 34568849
PLoS One. 2015 May 26;10(5):e0128240
pubmed: 26010904
Biosensors (Basel). 2017 Oct 31;7(4):
pubmed: 29088096
Anal Biochem. 2008 Jun 15;377(2):209-17
pubmed: 18405656
Annu Rev Pharmacol Toxicol. 2009;49:73-96
pubmed: 18834306
Nat Cell Biol. 2016 May 27;18(6):579-86
pubmed: 27230526
J Biol Chem. 2011 Jun 17;286(24):21277-86
pubmed: 21518764
Curr Opin Cell Biol. 2004 Apr;16(2):119-26
pubmed: 15196553
J Pharm Biomed Anal. 2013 Jan;72:150-4
pubmed: 23146240
Biochem J. 2017 Apr 13;474(9):1439-1451
pubmed: 28408429
J Cell Mol Med. 2008 Apr;12(2):363-73
pubmed: 18266959
BMC Struct Biol. 2008 May 16;8:26
pubmed: 18485199
Front Neurosci. 2017 Apr 06;11:185
pubmed: 28428740
Nat Protoc. 2013 Sep;8(9):1800-7
pubmed: 23989674
Mol Cell. 2005 Nov 23;20(4):525-38
pubmed: 16307917
Elife. 2019 Nov 12;8:
pubmed: 31713515
Elife. 2019 Dec 23;8:
pubmed: 31868589
Biochim Biophys Acta. 2014 Jan;1843(1):182-96
pubmed: 23850760
J Biol Chem. 2013 Jun 28;288(26):18939-46
pubmed: 23671280
Mol Cell. 2009 Feb 27;33(4):496-504
pubmed: 19250910
Comb Chem High Throughput Screen. 2009 Sep;12(8):791-800
pubmed: 19758119
J Pharm Biomed Anal. 2019 Sep 5;173:40-46
pubmed: 31108422
Neurochem Res. 1994 Apr;19(4):391-8
pubmed: 8065495
Biochem Pharmacol. 2014 Oct 1;91(3):293-300
pubmed: 25124703
J Biol Chem. 1990 Apr 15;265(11):6323-9
pubmed: 2156848
Cell Res. 2016 Apr;26(4):399-422
pubmed: 27012465
Structure. 2015 Mar 3;23(3):472-482
pubmed: 25684577
Viruses. 2021 Jun 21;13(6):
pubmed: 34205716
Arabidopsis Book. 2014 Oct 06;12:e0174
pubmed: 25320573
Cell Death Differ. 2011 Sep;18(9):1393-402
pubmed: 21372847
Anal Bioanal Chem. 2013 Mar;405(8):2535-44
pubmed: 23338757
J Biol Chem. 2001 Aug 17;276(33):31357-67
pubmed: 11397792
Nat Methods. 2004 Dec;1(3):203-8
pubmed: 15782195
Proc Natl Acad Sci U S A. 2003 Jul 8;100(14):8247-52
pubmed: 12821780
Front Chem. 2019 Dec 04;7:816
pubmed: 31867306
Nat Biotechnol. 2016 Mar;34(3):339-44
pubmed: 26789497
J Biol Chem. 1982 Sep 10;257(17):10329-37
pubmed: 6286650
Curr Protoc Protein Sci. 2015 Feb 02;79:19.25.1-19.25.26
pubmed: 25640894
Sci Adv. 2017 Jul 12;3(7):e1701620
pubmed: 28706995
PLoS One. 2022 Feb 2;17(2):e0263322
pubmed: 35108320
EMBO J. 2000 Jan 4;19(1):94-102
pubmed: 10619848
Biochem Biophys Res Commun. 2015 Oct 9;466(1):1-14
pubmed: 26325464
BMC Biol. 2018 Aug 10;16(1):88
pubmed: 30097011
Electrophoresis. 1999 Feb;20(2):418-28
pubmed: 10197449
Biochim Biophys Acta. 2004 Nov 29;1695(1-3):55-72
pubmed: 15571809
Proc Natl Acad Sci U S A. 2016 Aug 9;113(32):E4639-47
pubmed: 27385826
Methods Mol Biol. 2015;1278:165-82
pubmed: 25859949