A bead-based western for high-throughput cellular signal transduction analyses

Treindl, Fridolin; Ruprecht, Benjamin; Beiter, Yvonne; Schultz, S; Döttinger, Anette; Staebler, Annette; Joos, Thomas; Kling, Simon; Poetz, Oliver; Fehm, Tanja; Neubauer, Hans; Küster, Bernhard; Templin, Markus F.

doi:10.1038/ncomms12852

Cited by 88 publications

(119 citation statements)

References 33 publications

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“…Initially, 86 analytes were tested with our cohort using the DigiWest analysis tool (Supplementary Table 7) 33 . We extracted 49 analytes which displayed less than 10% background ( Supplementary Table 8…”

Section: Proteomic Analysis Revealed Differences In Mtor Pathway Compmentioning

confidence: 99%

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DNA methylation reveals distinct cells of origin for pancreatic neuroendocrine carcinomas (PanNECs) and pancreatic neuroendocrine tumors (PanNETs)

Simon

Riemer

Detjen

et al. 2020

Preprint

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Pancreatic Neuroendocrine Neoplasms (PanNENs) comprise a rare and heterogeneous group of tumors derived from neuroendocrine cells of the pancreas. Despite recent genetic and epigenetic characterization, biomarkers for improved patient stratification and personalized therapy are sparse and targeted therapies, including the mTOR inhibitor Everolimus, have shown limited success. To better define PanNENs tumors we performed multi-omic analyses on 59 tumors with varying grades (NET G1, NET G2, NET G3 and NEC), combining mutational profiling with epigenetic analysis and targeted proteomics. An unsupervised approach using DNA methylation profiles uncovered two major subgroups in PanNENs resembling α-like and β-like cells. DNA copy number analysis further divided the α-like subgroup into two distinct groups, indicating differential mechanisms of tumorigenesis from α-cells. β-like tumors however showed two distinct groups at the epigenetic level and suggest NET G3/NEC samples are of β-cell origin.DNA mutation profiling clearly separated α and β-cell derived PanNENs, whereby only αlike tumors had mutations within MEN1/DAXX/ATRX tumor suppressor genes. Targeted proteomic analysis further indicated overexpression of distinct components of the mTOR pathway. Our data provide further insights into the potential mechanisms behind PanNEN heterogeneity and form a basis for future diagnostic and therapy relevant patient stratification.

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Section: Proteomic Analysis Revealed Differences In Mtor Pathway Compmentioning

confidence: 99%

“…al. 33 , was used to identify antigen- Differentially expressed proteins between the groups were identified using student t-test and was corrected using Benjamini and Hochberg (BH) test in R. The identified candidates had an adjusted p-value < 0.05.…”

Section: Proteome Data Analysismentioning

confidence: 99%

DNA methylation reveals distinct cells of origin for pancreatic neuroendocrine carcinomas (PanNECs) and pancreatic neuroendocrine tumors (PanNETs)

Simon

Riemer

Detjen

et al. 2020

Preprint

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“…The DigiWest technology is a multistep process that begins with running samples on an SDS-PAGE gel and transferring to a nitrocellulose membrane [Treindl et al, 2016]. The DigiWest technology is a multistep process that begins with running samples on an SDS-PAGE gel and transferring to a nitrocellulose membrane [Treindl et al, 2016].…”

Section: Digiwestmentioning

confidence: 99%

“…Recent advances have extended the application of kinase capture on immobilized kinase inhibitors, with the power of Luminex immunodetection. The DigiWest technology is a multistep process that begins with running samples on an SDS-PAGE gel and transferring to a nitrocellulose membrane [Treindl et al, 2016]. The separated, membrane-bound proteins are then biotinylated, and each lane of the membrane is cut into 96 strips that cover a wide range of molecular weights.…”

Section: Digiwestmentioning

confidence: 99%

Measuring Kinase Activity—A Global Challenge

Cann

McDonald

East

et al. 2017

J of Cellular Biochemistry

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The kinase enzymes within a cell, known collectively as the kinome, play crucial roles in many signaling pathways, including survival, motility, differentiation, stress response, and many more. Aberrant signaling through kinase pathways is often linked to cancer, among other diseases. A major area of scientific research involves understanding the relationships between kinases, their targets, and how the kinome adapts to perturbations of the cellular system. This review will discuss many of the current and developing methods for studying kinase activity, and evaluate their applications, advantages, and disadvantages. J. Cell. Biochem. 118: 3595-3606, 2017. © 2017 Wiley Periodicals, Inc.

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“…Over the recent decades, bead-based assay methodologies have had a noticeable impact on the detection of biological and chemical analytes12. The surface of a microbead can be functionalized using various receptors (e.g., antibodies, peptides, DNA, mRNA, enzymes) and their compounds.…”

mentioning

confidence: 99%

Deterministic bead-in-droplet ejection utilizing an integrated plug-in bead dispenser for single bead–based applications

Kim

Choi

Lee

et al. 2017

Sci Rep

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This paper presents a deterministic bead-in-droplet ejection (BIDE) technique that regulates the precise distribution of microbeads in an ejected droplet. The deterministic BIDE was realized through the effective integration of a microfluidic single-particle handling technique with a liquid dispensing system. The integrated bead dispenser facilitates the transfer of the desired number of beads into a dispensing volume and the on-demand ejection of bead-encapsulated droplets. Single bead–encapsulated droplets were ejected every 3 s without any failure. Multiple-bead dispensing with deterministic control of the number of beads was demonstrated to emphasize the originality and quality of the proposed dispensing technique. The dispenser was mounted using a plug-socket type connection, and the dispensing process was completely automated using a programmed sequence without any microscopic observation. To demonstrate a potential application of the technique, bead-based streptavidin–biotin binding assay in an evaporating droplet was conducted using ultralow numbers of beads. The results evidenced the number of beads in the droplet crucially influences the reliability of the assay. Therefore, the proposed deterministic bead-in-droplet technology can be utilized to deliver desired beads onto a reaction site, particularly to reliably and efficiently enrich and detect target biomolecules.

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A bead-based western for high-throughput cellular signal transduction analyses

Cited by 88 publications

References 33 publications

DNA methylation reveals distinct cells of origin for pancreatic neuroendocrine carcinomas (PanNECs) and pancreatic neuroendocrine tumors (PanNETs)

DNA methylation reveals distinct cells of origin for pancreatic neuroendocrine carcinomas (PanNECs) and pancreatic neuroendocrine tumors (PanNETs)

Measuring Kinase Activity—A Global Challenge

Deterministic bead-in-droplet ejection utilizing an integrated plug-in bead dispenser for single bead–based applications

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