Exploring blocking assays using Octet, ProteOn, and Biacore biosensors

Abdiche, Yasmina; Malashock, Dan; Pinkerton, Alanna; Pons, Jaume

doi:10.1016/j.ab.2008.11.038

Cited by 81 publications

(62 citation statements)

References 16 publications

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“…Mitochondrial Thiolase Directly Interacts with p46Shc with 55 nM K D -We determined that p46Shc and thiolase directly interact in vitro using BLI to detect real-time protein-protein interactions (12)(13)(14). We expressed in HEK cells and purified biotinylated ACAA2, p46Shc, p46Shc␦SH2 (p46Shc lacking the SH2 domain), and GFP as a non-binding control (Fig.…”

Section: P46shc Is the Only Mitochondrial Shc Isoform Under Physiologmentioning

confidence: 99%

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p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria

Tomilov

Tomilova

Shan

et al. 2016

Journal of Biological Chemistry

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Although the p46Shc isoform has been known to be mitochondrially localized for 11 years, its function in mitochondria has been a mystery. We confirmed p46Shc to be mitochondrially localized and showed that the major mitochondrial partner of p46Shc is the lipid oxidation enzyme 3-ketoacylCoA thiolase ACAA2, to which p46Shc binds directly and with a strong affinity. Increasing p46Shc expression inhibits, and decreasing p46Shc stimulates enzymatic activity of thiolase in vitro. Thus, we suggest p46Shc to be a negative mitochondrial thiolase activity regulator, and reduction of p46Shc expression activates thiolase. This is the first demonstration of a protein that directly binds and controls thiolase activity. Thiolase was thought previously only to be regulated by metabolite balance and steadystate flux control. Thiolase is the last enzyme of the mitochondrial fatty acid beta-oxidation spiral, and thus is important for energy metabolism. Mice with reduction of p46Shc are lean, resist obesity, have higher lipid oxidation capacity, and increased thiolase activity. The thiolase-p46Shc connection shown here in vitro and in organello may be an important underlying mechanism explaining the metabolic phenotype of Shcdepleted mice in vivo.Shc proteins have three isoforms: p46, p52, and p66, and have major effects on metabolism (1-4). p52Shc contacts the insulin receptor and regulates the signaling between the IRS1 and Ras pathway (5). In 2004, it was demonstrated that the major mitochondrial Shc isoform is p46Shc (6), but since that time the mitochondrial partner and physiological function of p46Shc has been a mystery. Here we show the mitochondrial partner and function of p46Shc, and suggest how this could contribute to the obesity-resistance observed in ShcKO mice.There are three isoforms at the mammalian Shc locus, the highly expressed isoforms p46Shc and p52Shc, and the minor p66Shc. All three Shc isoforms are derived from a single DNA locus. First, two mRNA are produced: p66Shc and p52/46Shc by means of trans-splicing. The p66Shc mRNA has start codons for all three Shc isoforms. The p52/46Shc-mRNA does not have a start codon for p66Shc and only produces p52Shc and p46Shc (7). For this reason, knockdowns of either p66Shc, or all three Shc isoforms together have been achieved to date. There are two mouse models of Shc depletion produced to date: ShcL, also known as p66ShcKO developed by the Tom Prolla group (ShcProlla, Ref. 4), and Shc mice developed by the Pelicci group (ShcP or ShcPelicci, also known as p66Shc (Ϫ/Ϫ, Refs. 3,4,8,9). Names in literature for these two models can be described by these definitions: ShcL, ShcProlla; ShcKO, ShcP ϭ ShcPelicci ϭ p66Shc(Ϫ/Ϫ).Briefly, ShcL or ShcProlla mice have a deletion of only the minor p66Shc isoform and have no health benefits: ShcL mice are not lean, do not resist weight gain on high fat diets (HFD), 2 and are not longer-lived on HFD, and do not have improved insulin sensitivity (4). Thus p66Shc deletion by itself does not result in health benefits.By contrast, ShcKO have...

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Section: P46shc Is the Only Mitochondrial Shc Isoform Under Physiologmentioning

confidence: 99%

“…We carried out mass spectrometry, two-dimensional gel electrophoresis, and bio-layer interferometry (BLI) (12)(13)(14) assays to identify the major mitochondrial partner of p46Shc. These assays identified mitochondrial 3-ketoacylCoA thiolase (ACAA2) as the p46Shc major partner.…”

mentioning

confidence: 99%

p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria

Tomilov

Tomilova

Shan

et al. 2016

Journal of Biological Chemistry

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“…[20][21][22] Here, we describe a high throughput method to detect antibody clone self-interaction by bio-layer interferometry (CSI-BLI) with low material consumption.…”

Section: High Throughput Detection Of Antibody Self-interaction By Bimentioning

confidence: 99%

High throughput detection of antibody self-interaction by bio-layer interferometry

Sun

Reid

Liu

et al. 2013

mAbs

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“…The ForteBio Octet uses similar white light spectroscopy (21)(22)(23) ), but appears to be a simplification in that it interrogates directly internal reflection from the tip of a probe at the end of an optical fiber. The incident light and reflected light interfere, and the interference is affected by the optical thickness of the biological layer on the probe tip (biolayer interferometry).…”

Section: Optical Immunosensor Systemsmentioning

confidence: 99%

Discerning Trends in Multiplex Immunoassay Technology with Potential for Resource-Limited Settings

Gordon

Michel

2012

Clinical Chemistry

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BACKGROUND In the search for more powerful tools for diagnoses of endemic diseases in resource-limited settings, we have been analyzing technologies with potential applicability. Increasingly, the process focuses on readily accessible bodily fluids combined with increasingly powerful multiplex capabilities to unambiguously diagnose a condition without resorting to reliance on a sophisticated reference laboratory. Although these technological advances may well have important implications for the sensitive and specific detection of disease, to date their clinical utility has not been demonstrated, especially in resource-limited settings. Furthermore, many emerging technological developments are in fields of physics or engineering, which are not readily available to or intelligible to clinicians or clinical laboratory scientists. CONTENT This review provides a look at technology trends that could have applicability to high-sensitivity multiplexed immunoassays in resource-limited settings. Various technologies are explained and assessed according to potential for reaching relevant limits of cost, sensitivity, and multiplex capability. Frequently, such work is reported in technical journals not normally read by clinical scientists, and the authors make enthusiastic claims for the potential of their technology while ignoring potential pitfalls. Thus it is important to draw attention to technical hurdles that authors may not be publicizing. SUMMARY Immunochromatographic assays, optical methods including those involving waveguides, electrochemical methods, magnetorestrictive methods, and field-effect transistor methods based on nanotubes, nanowires, and nanoribbons reveal possibilities as next-generation technologies.

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Exploring blocking assays using Octet, ProteOn, and Biacore biosensors

Cited by 81 publications

References 16 publications

p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria

p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria

High throughput detection of antibody self-interaction by bio-layer interferometry

Discerning Trends in Multiplex Immunoassay Technology with Potential for Resource-Limited Settings

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