Antibody validation for Western blot: By the user, for the user.
Antibodies
/ metabolism
Antibody Specificity
/ immunology
Blotting, Western
Cell Line, Tumor
Epitopes
/ immunology
Fluorescence
Humans
Immunoblotting
Peer Review, Research
Peptides
/ metabolism
Phosphorylation
Poly(ADP-ribose) Polymerases
/ metabolism
Protein Processing, Post-Translational
Reproducibility of Results
Immunoblot
Methodology
antibody
antibody validation
antigen
immunochemistry
irreproducibility crisis
protein domain
reproducibility
western blot
Journal
The Journal of biological chemistry
ISSN: 1083-351X
Titre abrégé: J Biol Chem
Pays: United States
ID NLM: 2985121R
Informations de publication
Date de publication:
24 01 2020
24 01 2020
Historique:
received:
06
08
2019
revised:
20
11
2019
pubmed:
11
12
2019
medline:
5
9
2020
entrez:
11
12
2019
Statut:
ppublish
Résumé
Well-characterized antibody reagents play a key role in the reproducibility of research findings, and inconsistent antibody performance leads to variability in Western blotting and other immunoassays. The current lack of clear, accepted standards for antibody validation and reporting of experimental details contributes to this problem. Because the performance of primary antibodies is strongly influenced by assay context, recommendations for validation and usage are unique to each type of immunoassay. Practical strategies are proposed for the validation of primary antibody specificity, selectivity, and reproducibility using Western blot analysis. The antibody should produce reproducible results within and between Western blotting experiments and the observed effect confirmed with a complementary or orthogonal method. Routine implementation of standardized antibody validation and reporting in immunoassays such as Western blotting may promote improved reproducibility across the global life sciences community.
Identifiants
pubmed: 31819006
pii: S0021-9258(17)49905-4
doi: 10.1074/jbc.RA119.010472
pmc: PMC6983856
doi:
Substances chimiques
Antibodies
0
Epitopes
0
Peptides
0
Poly(ADP-ribose) Polymerases
EC 2.4.2.30
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
926-939Informations de copyright
© 2020 Pillai-Kastoori et al.
Références
Am J Physiol Cell Physiol. 2015 Mar 15;308(6):C426-33
pubmed: 25540176
Cell. 2016 Apr 21;165(3):535-50
pubmed: 27104977
Genome Biol. 2003;4(9):117
pubmed: 12952525
J Histochem Cytochem. 2009 Jan;57(1):7-8
pubmed: 18854593
Anal Methods. 2016 Oct 21;8(39):7002-7013
pubmed: 28392839
Neuron. 2016 May 4;90(3):434-6
pubmed: 27151636
J Clin Pathol. 2004 Sep;57(9):912-7
pubmed: 15333649
Front Immunol. 2013 Jul 29;4:217
pubmed: 23908655
Cancer Res. 2014 Aug 1;74(15):4024-9
pubmed: 25035389
J Histochem Cytochem. 2014 Oct;62(10):693-7
pubmed: 25023613
Mol Cell Proteomics. 2010 Jan;9(1):1-10
pubmed: 19674966
mBio. 2016 Aug 30;7(4):
pubmed: 27578756
F1000Res. 2017 Jun 8;6:851
pubmed: 28713558
Biochemistry. 2013 Jun 4;52(22):3797-806
pubmed: 23517332
Nat Methods. 2018 Nov;15(11):909-912
pubmed: 30377371
mBio. 2016 Nov 8;7(6):
pubmed: 27834205
Electrophoresis. 2005 Jun;26(12):2470-5
pubmed: 15924365
Nature. 2015 Nov 26;527(7579):545-51
pubmed: 26607547
J Biol Chem. 2015 Dec 11;290(50):29692-4
pubmed: 26657753
Biotechniques. 2010 Mar;48(3):197-209
pubmed: 20359301
Nature. 2016 May 25;533(7604):437
pubmed: 27225078
Science. 2014 Jan 3;343(6166):84-87
pubmed: 24336571
Nature. 2017 Mar 15;543(7645):288
pubmed: 28300127
Nat Methods. 2005 Dec;2(12):905-9
pubmed: 16299475
Nat Methods. 2016 Oct;13(10):823-7
pubmed: 27595404
Cell. 2013 May 9;153(4):910-8
pubmed: 23643243
J Clin Invest. 2009 Jun;119(6):1714-26
pubmed: 19451695
J Histochem Cytochem. 2009 Jan;57(1):1-5
pubmed: 18854594
Curr Protoc Protein Sci. 2013 Sep 24;73:9.9.1-9.9.23
pubmed: 24510596
Proteomics. 2008 Jun;8(12):2379-83
pubmed: 18563731
Sci Signal. 2015 Apr 07;8(371):fs7
pubmed: 25852186
Expert Rev Proteomics. 2014 Oct;11(5):549-60
pubmed: 25059473
Anal Chim Acta. 2012 Jan 4;709:21-31
pubmed: 22122927
N Am J Med Sci. 2012 Sep;4(9):429-34
pubmed: 23050259
Nat Methods. 2017 Feb 28;14(3):215-216
pubmed: 28245211
Scand J Med Sci Sports. 2017 Jan;27(1):4-25
pubmed: 27263489
Nature. 2017 Aug 22;548(7668):387-388
pubmed: 28836615
J Comp Neurol. 2005 Dec 26;493(4):477-8
pubmed: 16304632
Anal Biochem. 1989 Sep;181(2):318-20
pubmed: 2817396
Anal Chem. 2010 May 15;82(10):3974-6
pubmed: 20426486
J Histochem Cytochem. 2012 Mar;60(3):174-87
pubmed: 22215633
Mt Sinai J Med. 2006 Jul;73(4):673-81
pubmed: 16878273
Nature. 2016 May 25;533(7604):452-4
pubmed: 27225100
Nat Commun. 2018 Oct 8;9(1):4130
pubmed: 30297845
Mol Neurodegener. 2010 Nov 05;5:48
pubmed: 21054848
Nat Methods. 2017 Feb 28;14(3):215
pubmed: 28245212
mBio. 2018 Feb 20;9(1):
pubmed: 29463650
Neuroscience. 2005;136(3):649-60
pubmed: 16344142
Nature. 2014 Jan 30;505(7485):612-3
pubmed: 24482835
Biochim Biophys Acta. 2015 Dec;1856(2):234-43
pubmed: 26434948
J Histochem Cytochem. 1999 Oct;47(10):1233-6
pubmed: 10490451
Public Health Rep. 2002 Nov-Dec;117(6):521-33
pubmed: 12576532
Nature. 2017 May 31;546(7656):8
pubmed: 28569823
Methods Mol Biol. 2015;1314:27-32
pubmed: 26139251
J Biomech. 2017 Mar 21;54:1-3
pubmed: 28274476
J Comp Neurol. 2003 Oct 13;465(2):161-3
pubmed: 12949777
Oncoscience. 2015 May 14;2(5):542-54
pubmed: 26097887
Endocrinology. 2014 Mar;155(3):676-87
pubmed: 24428532
Cold Spring Harb Protoc. 2010 May;2010(5):pdb.prot5421
pubmed: 20439408
Mol Biochem Parasitol. 2001 Aug;116(1):55-63
pubmed: 11463466
J Immunol Methods. 1989 Aug 15;122(1):129-35
pubmed: 2760474
Dev Biol Stand. 1997;91:3-13
pubmed: 9413677
Nat Biotechnol. 2010 Jul;28(7):650-3
pubmed: 20622827
Anal Chem Insights. 2018 Feb 08;13:1177390118757462
pubmed: 29467569
Nat Methods. 2014 Sep;11(9):895-8
pubmed: 25166868
Nat Biotechnol. 2008 Mar;26(3):317-25
pubmed: 18278033
Sci Signal. 2015 Apr 07;8(371):rs2
pubmed: 25852189
Anal Biochem. 1985 Oct;150(1):76-85
pubmed: 3843705
Curr Biol. 1993 Jun 1;3(6):327-32
pubmed: 15335725
Naunyn Schmiedebergs Arch Pharmacol. 2009 Apr;379(4):413-5
pubmed: 19096766
Science. 2012 Aug 17;337(6096):816-21
pubmed: 22745249
Anal Chem. 2016 Jul 5;88(13):6703-10
pubmed: 27270033
F1000Res. 2016 Mar 09;5:308
pubmed: 26998240
PLoS One. 2015 Aug 19;10(8):e0135392
pubmed: 26287535
Sci Rep. 2017 Aug 31;7(1):10300
pubmed: 28860483
Biochim Biophys Acta. 2016 Feb;1863(2):284-92
pubmed: 26646257
Cell. 2014 Jun 5;157(6):1262-78
pubmed: 24906146
Curr Protoc Cell Biol. 2005 Jul;Chapter 21:21.4.1-21.4.13
pubmed: 18228466
F1000Res. 2017 May 2;6:604
pubmed: 28620458
Front Neurol. 2015 Aug 19;6:179
pubmed: 26347708