Reproducibility: Standardize antibodies used in research

Bradbury, Andrew; Plückthun, Andreas

doi:10.1038/518027a

Cited by 558 publications

(488 citation statements)

References 11 publications

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“…Antibody quality in terms of target specificity and reproducibility is one of the emerging issues in the biological sciences [36,37]. In this study, we generated a monoclonal antibody that specifically detects diacetylation at K5 and K8 of histone H4, a hallmark of H4-hyperacetylated chromatin states in vivo .…”

Section: Discussionmentioning

confidence: 99%

JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states

Handoko¹,

Kaczkowski²,

Hon³

et al. 2018

Epigenetics

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The bromodomain and extra-terminal domain (BET) proteins are promising drug targets for cancer and immune diseases. However, BET inhibition effects have been studied more in the context of bromodomain-containing protein 4 (BRD4) than BRD2, and the BET protein association to histone H4-hyperacetylated chromatin is not understood at the genome-wide level. Here, we report transcription start site (TSS)-resolution integrative analyses of ChIP-seq and transcriptome profiles in human non-small cell lung cancer (NSCLC) cell line H23. We show that di-acetylation at K5 and K8 of histone H4 (H4K5acK8ac) co-localizes with H3K27ac and BRD2 in the majority of active enhancers and promoters, where BRD2 has a stronger association with H4K5acK8ac than H3K27ac. Although BET inhibition by JQ1 led to complete reduction of BRD2 binding to chromatin, only local changes of H4K5acK8ac levels were observed, suggesting that recruitment of BRD2 does not influence global histone H4 hyperacetylation levels. This finding supports a model in which recruitment of BET proteins via histone H4 hyperacetylation is predominant over hyperacetylation of histone H4 by BET protein-associated acetyltransferases. In addition, we found that a remarkable number of BRD2-bound genes, including MYC and its downstream target genes, were transcriptionally upregulated upon JQ1 treatment. Using BRD2-enriched sites and transcriptional activity analysis, we identified candidate transcription factors potentially involved in the JQ1 response in BRD2-dependent and -independent manner.

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Section: Discussionmentioning

confidence: 99%

JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states

Handoko¹,

Kaczkowski²,

Hon³

et al. 2018

Epigenetics

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“…The waste, in terms of time and resources, is colossal. Losses from purchasing poorly characterized antibodies have been estimated at $800 million per year, not counting the impact of false conclusions, uninterpretable (or misinterpreted) experiments, wasted patient samples and fruitless research time 2 . Mathias Uhlén, a protein researcher at the Royal Institute of Technology in Stockholm, says that frustration with research antibodies has been building for years 3 and that the time is finally ripe for improvements.…”

mentioning

confidence: 99%

Antibody anarchy: A call to order

Baker¹

2015

Nature

121

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“…Moreover, polyclonal antibodies usually exhibit affinity towards several epitopes. It has been claimed that only 0.5-5% of the antibodies in a polyclonal reagent bind to their intended target [25].…”

Section: Discussionmentioning

confidence: 99%

“…The recommended applications are mainly immunohistochemistry (IHC) or immunocytochemistry (ICC), but also western blot, EIA and ELISA. Recently, several studies have pointed out the quality issues and traceability problem concerning commercial antibodies and the lack of quality in the antibody market has been widely recognized by the scientific community [19][20][21][22][23][24][25][26][27][28]. Often, the characterization and specifications provided by the manufacturers are somewhat incomplete and, furthermore, the authors often fail to identify the exact antibody used [28][29][30] for other researchers to be able to purchase the same product.…”

Section: Commercial Anti-histamine Antibodies and Their Use In Immunomentioning

confidence: 99%

Challenges in Developing a Biochip for Intact Histamine Using Commercial Antibodies

2017

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This study describes the development and the challenges in the development of an on-chip immunoassay for histamine using commercially available antibodies. Histamine can be used as an indicator of food freshness and quality, but it is also a relevant marker in clinical diagnostics. Due to its low molecular weight, simple structure and thus low immunogenicity production of high specificity and affinity antibodies is difficult. From six commercial anti-histamine antibodies tested, only two bound the histamine free in the solution. A fluorescent on-chip immunoassay for histamine was established with a dynamic range of 8-111 µg/mL using polyclonal anti-histamine antibody H7403 from Sigma (Mendota Heights, MN, USA). The anti-histamine antibodies described and used in published literature are thoroughly reviewed and the quality of commercial antibodies and their traceability and quality issues are highlighted and extensively discussed.

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Reproducibility: Standardize antibodies used in research

Cited by 558 publications

References 11 publications

JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states

JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states

Antibody anarchy: A call to order

Challenges in Developing a Biochip for Intact Histamine Using Commercial Antibodies

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