The Experts below are selected from a list of 30198 Experts worldwide ranked by ideXlab platform
Joshua Labaer - One of the best experts on this subject based on the ideXlab platform.
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quantifying antibody binding on Protein Microarrays using microarray nonlinear calibration
BioTechniques, 2013Co-Authors: Garrick Wallstrom, Jie Wang, Xiaofang Bian, Dewey Mitchell Magee, Eliseo D A Mendoza, Morgan Graves, Ji Qiu, Joshua LabaerAbstract:We present a microarray nonlinear calibration (MiNC) method for quantifying antibody binding to the surface of Protein Microarrays that significantly increases the linear dynamic range and reduces assay variation compared with traditional approaches. A serological analysis of guinea pig Mycobacterium tuberculosis models showed that a larger number of putative antigen targets were identified with MiNC, which is consistent with the improved assay performance of Protein Microarrays. MiNC has the potential to be employed in biomedical research using multiplex antibody assays that need quantitation, including the discovery of antibody biomarkers, clinical diagnostics with multi-antibody signatures, and construction of immune mathematical models.
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quantifying antibody binding on Protein Microarrays using microarray nonlinear calibration
BioTechniques, 2013Co-Authors: Xiaobo Yu, Jie Wang, Garrick Wallstrom, Xiaofang Bian, Dewey Mitchell Magee, Eliseo D A Mendoza, Morgan Graves, Joshua LabaerAbstract:We present a microarray nonlinear calibration (MiNC) method for quantifying antibody binding to the surface of Protein Microarrays that significantly increases the linear dynamic range and reduces ...
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Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 4: DNA biotinylation, precipitation, and arraying of samples
Cold Spring Harbor Protocols, 2008Co-Authors: Andrew J Link, Joshua LabaerAbstract:INTRODUCTIONFunctional proteomics enables Protein activities to be studied in vitro using high-throughput (HT) methods. Protein Microarrays are the method of choice because they display many Proteins simultaneously and require only small reaction volumes to assess function. Protein Microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many Proteins spotted on the array. Target Protein Microarrays are usually generated by expressing, purifying, and spotting the Proteins onto a solid surface at very close spatial density. An alternative approach is to translate the Proteins in situ on the array surface. This method uses cell-free extracts that transcribe and translate DNA into Proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target Proteins. Instead of printing Proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired Proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of Proteins and DNA using antibodies and stains. This protocol describes DNA biotinylation, precipitation, and arraying in preparation for Protein expression.
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Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 1: Coating glass slides with amino silane
Cold Spring Harbor Protocols, 2008Co-Authors: Andrew J Link, Joshua LabaerAbstract:INTRODUCTIONFunctional proteomics enables Protein activities to be studied in vitro using high-throughput (HT) methods. Protein Microarrays are the method of choice because they display many Proteins simultaneously and require only small reaction volumes to assess function. Protein Microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many Proteins spotted on the array. Target Protein Microarrays are usually generated by expressing, purifying, and spotting the Proteins onto a solid surface at very close spatial density. An alternative approach is to translate the Proteins in situ on the array surface. This approach, termed "Nucleic Acid Protein Programmable Array" (NAPPA), enables the simultaneous expression of Proteins in microarray format without the need for individual Protein purification. This method uses cell-free extracts that transcribe and translate DNA into Proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target Proteins. Instead of printing Proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired Proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of Proteins and DNA using antibodies and stains. This protocol describes the initial preparation of slides to be used in the method.
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Applications of Protein Microarrays for biomarker discovery
Proteomics - Clinical Applications, 2008Co-Authors: Niroshan Ramachandran, Sanjeeva Srivastava, Joshua LabaerAbstract:The search for new biomarkers for diagnosis, prognosis, and therapeutic monitoring of diseases continues in earnest despite dwindling success at finding novel reliable markers. Some of the current markers in clinical use do not provide optimal sensitivity and specificity, with the prostate cancer antigen (PSA) being one of many such examples. The emergence of proteomic techniques and systems approaches to study disease pathophysiology has rekindled the quest for new biomarkers. In particular the use of Protein Microarrays has surged as a powerful tool for large-scale testing of biological samples. Approximately half the reports on Protein Microarrays have been published in the last two years especially in the area of biomarker discovery. In this review, we will discuss the application of Protein microarray technologies that offer unique opportunities to find novel biomarkers.
Sanjeeva Srivastava - One of the best experts on this subject based on the ideXlab platform.
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Protein microarray applications autoantibody detection and posttranslational modification
Proteomics, 2016Co-Authors: Apurva Atak, Shuvolina Mukherjee, Rekha Jain, Shabarni Gupta, Vedita Anand Singh, Nikita Gahoi, K P Manubhai, Sanjeeva SrivastavaAbstract:The discovery of DNA Microarrays was a major milestone in genomics; however, it could not adequately predict the structure or dynamics of underlying Protein entities, which are the ultimate effector molecules in a cell. Protein Microarrays allow simultaneous study of thousands of Proteins/peptides, and various advancements in array technologies have made this platform suitable for several diagnostic and functional studies. Antibody arrays enable researchers to quantify the abundance of target Proteins in biological fluids and assess PTMs by using the antibodies. Protein Microarrays have been used to assess Protein-Protein interactions, Protein-ligand interactions, and autoantibody profiling in various disease conditions. Here, we summarize different microarray platforms with focus on its biological and clinical applications in autoantibody profiling and PTM studies. We also enumerate the potential of tissue Microarrays to validate findings from Protein arrays as well as other approaches, highlighting their significance in proteomics.
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Protein Microarrays and novel detection platforms
Expert Review of Proteomics, 2011Co-Authors: Harini Chandra, Panga Jaipal Reddy, Sanjeeva SrivastavaAbstract:The field of proteomics has undergone rapid advancements over the last decade and Protein Microarrays have emerged as a promising technological platform for the challenging task of studying complex proteomes. This gel-free approach has found an increasing number of applications due to its ability to rapidly and efficiently study thousands of Proteins simultaneously. Different Protein Microarrays, including capture arrays, reverse-phase arrays, tissue Microarrays, lectin Microarrays and cell-free expression Microarrays, have emerged, which have demonstrated numerous applications for proteomics studies including biomarker discovery, Protein interaction studies, enzyme-substrate profiling, immunological profiling and vaccine development, among many others. The need to detect extremely low-abundance Proteins in complex mixtures has provided motivation for the development of sensitive, real-time and multiplexed detection platforms. Conventional label-based approaches like fluorescence, chemiluminescence and use of radioactive isotopes have witnessed substantial advancements, with techniques like quantum dots, gold nanoparticles, dye-doped nanoparticles and several bead-based methods now being employed for Protein microarray studies. In order to overcome the limitations posed by label-based technologies, several label-free approaches like surface plasmon resonance, carbon nanotubes and nanowires, and microcantilevers, among others, have also advanced in recent years, and these methods detect the query molecule itself. The scope of this article is to outline the Protein microarray techniques that are currently being used for analytical and function-based proteomics and to provide a detailed analysis of the key technological advances and applications of various detection systems that are commonly used with Microarrays.
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label free detection techniques for Protein Microarrays prospects merits and challenges
Proteomics, 2010Co-Authors: Sandipan Ray, Gunjan D Mehta, Sanjeeva SrivastavaAbstract:Protein Microarrays, on which thousands of discrete Proteins are printed, provide a valuable platform for functional analysis of the proteome. They have been widely used for biomarker discovery and to study Protein-Protein interactions. The accomplishments of DNA microarray technology, which had enabled massive parallel studies of gene expression, sparked great interest for the development of Protein Microarrays to achieve similar success at the Protein level. Protein microarray detection techniques are often classified as being label-based and label-free. Most of the microarray applications have employed labelled detection such as fluorescent, chemiluminescent and radioactive labelling. These labelling strategies have synthetic challenges, multiple label issues and may exhibit interference with the binding site. Therefore, development of sensitive, reliable, high-throughput, label-free detection techniques are now attracting significant attention. Label-free detection techniques monitor biomolecular interactions and simplify the bioassays by eliminating the need for secondary reactants. Moreover, they provide quantitative information for the binding kinetics. In this article, we will review several label-free techniques, which offer promising applications for the Protein Microarrays, and discuss their prospects, merits and challenges.
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Applications of Protein Microarrays for biomarker discovery
Proteomics - Clinical Applications, 2008Co-Authors: Niroshan Ramachandran, Sanjeeva Srivastava, Joshua LabaerAbstract:The search for new biomarkers for diagnosis, prognosis, and therapeutic monitoring of diseases continues in earnest despite dwindling success at finding novel reliable markers. Some of the current markers in clinical use do not provide optimal sensitivity and specificity, with the prostate cancer antigen (PSA) being one of many such examples. The emergence of proteomic techniques and systems approaches to study disease pathophysiology has rekindled the quest for new biomarkers. In particular the use of Protein Microarrays has surged as a powerful tool for large-scale testing of biological samples. Approximately half the reports on Protein Microarrays have been published in the last two years especially in the area of biomarker discovery. In this review, we will discuss the application of Protein microarray technologies that offer unique opportunities to find novel biomarkers.
Lance A Liotta - One of the best experts on this subject based on the ideXlab platform.
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combining the sibling technologies of laser capture microdissection and reverse phase Protein Microarrays
Advances in Experimental Medicine and Biology, 2019Co-Authors: Claudius Mueller, Justin B Davis, Lance A LiottaAbstract:Reverse phase Protein Microarrays (RPPA) and laser capture microdissection (LCM) are “sibling” technologies that originated from the same laboratory to overcome the challenge of quantifying low-abundance Proteins in heterogeneous tissues. Combining both technologies provides both unique opportunities and unique challenges. Enabling the unprecedented resolution of the activation state of labile biomarkers, such as phosphorylated cell signaling Proteins, has had a substantial impact on our understanding of diseases and is playing a significant role in clinical trials. At the same time, quantifying Proteins at this sensitivity in very small amounts of material requires cognizance of pre-analytical variability and the limits of downstream detection technologies. Here, we discuss both the potential that the combination of both technologies presents and the potential pitfalls that must be navigated.
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reverse phase Protein Microarrays advance to use in clinical trials
Molecular Oncology, 2010Co-Authors: Claudius Mueller, Lance A Liotta, Virginia EspinaAbstract:Individualizing cancer therapy for molecular targeted inhibitors requires a new class of molecular profiling technology that can map the functional state of the cancer cell signal pathways containing the drug targets. Reverse phase Protein Microarrays (RPMA) are a technology platform designed for quantitative, multiplexed analysis of specific phosphorylated, cleaved, or total (phosphorylated and non-phosphorylated) forms of cellular Proteins from a limited amount of sample. This class of microarray can be used to interrogate tissue samples, cells, serum, or body fluids. RPMA were previously a research tool; now this technology has graduated to use in research clinical trials with clinical grade sensitivity and precision. In this review we describe the application of RPMA for multiplexed signal pathway analysis in therapeutic monitoring, biomarker discovery, and evaluation of pharmaceutical targets, and conclude with a summary of the technical aspects of RPMA construction and analysis.
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reverse phase Protein Microarrays application to biomarker discovery and translational medicine
Expert Review of Molecular Diagnostics, 2007Co-Authors: Amy Vanmeter, Virginia Espina, Lance A Liotta, Michele Signore, Mariaelena Pierobon, Emanuel F PetricoinAbstract:Mapping of Protein signaling networks within tumors can identify new targets for therapy and provide a means to stratify patients for individualized therapy. Kinases are important drug targets, as such kinase network information could become the basis for development of therapeutic strategies for improving treatment outcome. An urgent clinical goal is to identify functionally important molecular networks associated with subpopulations of patients that may not respond to conventional combination chemotherapy. Reverse-phase Protein Microarrays are a technology platform designed for quantitative, multiplexed analysis of specific phosphorylated, cleaved, or total (phosphorylated and nonphosphorylated) forms of cellular Proteins from a limited amount of sample. This class of microarray can be used to interrogate cellular samples, serum or body fluids. This review focuses on the application of reverse-phase Protein Microarrays for translational research and therapeutic drug target discovery.
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physicochemically modified silicon as a substrate for Protein Microarrays
Biomaterials, 2007Co-Authors: Jasper A Nijdam, Virginia Espina, Emanuel F Petricoin, David Geho, Mark Mingcheng Cheng, Roberta Fedele, Paul C Herrmann, Keith Killian, Lance A LiottaAbstract:Reverse phase Protein Microarrays (RPMA) enable high throughput screening of posttranslational modifications of important signaling Proteins within diseased cells. One limitation of Protein-based molecular profiling is the lack of a PCR-like intrinsic amplification system for Proteins. Enhancement of Protein microarray sensitivities is an important goal, especially because many molecular targets within patient tissues are of low abundance. The ideal array substrate will have a high Protein-binding affinity and low intrinsic signal. To date, nitrocellulose-coated glass has provided an effective substrate for Protein binding in the microarray format when using chromogenic detection systems. As fluorescent systems, such as quantum dots, are explored as potential reporter agents, the intrinsic fluorescent properties of nitrocellulose-coated glass slides limit the ability to image Microarrays for extended periods of time where increases in net sensitivity can be attained. Silicon, with low intrinsic autofluorescence, is being explored as a potential microarray surface. Native silicon has low binding potential. Through titrated reactive ion etching (RIE), varying surface areas have been created on silicon in order to enhance Protein binding. Further, via chemical modification, reactive groups have been added to the surfaces for comparison of relative Protein binding. Using this combinatorial method of surface roughening and surface coating, 3-aminopropyltriethoxysilane (APTES) and mercaptopropyltrimethoxysilane (MPTMS) treatments were shown to transform native silicon into a Protein-binding substrate comparable to nitrocellulose.
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Protein microarray detection strategies focus on direct detection technologies
Journal of Immunological Methods, 2004Co-Authors: Virginia Espina, Elisa C Woodhouse, Julia Wulfkuhle, Heather D Asmussen, Emanuel F Petricoin, Lance A LiottaAbstract:Protein Microarrays are being utilized for functional proteomic analysis, providing information not obtainable by gene arrays. Microarray technology is applicable for studying Protein-Protein, Protein-ligand, kinase activity and posttranslational modifications of Proteins. A precise and sensitive Protein microarray, the direct detection or reverse-phase microarray, has been applied to ongoing clinical trials at the National Cancer Institute for studying phosphorylation events in EGF-receptor-mediated cell signaling pathways. The variety of microarray applications allows for multiple, creative microarray designs and detection strategies. Herein, we discuss detection strategies and challenges for Protein microarray technology, focusing on direct detection of Protein Microarrays.
Emanuel F Petricoin - One of the best experts on this subject based on the ideXlab platform.
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abstract p2 04 02 identifying molecular targets and mechanisms of treatment resistance in inflammatory breast cancer ibc using reverse phase Protein Microarrays rpma
Cancer Research, 2015Co-Authors: Laura Austin, Emanuel F Petricoin, Kimberly Limentani, Juan P Palazzo, T Avery, Rebecca Jaslow, Ron Hencin, Massimo CristofanilliAbstract:Background Inflammatory Breast Cancer (IBC) is a clinicopathologic diagnosis characterized by rapid progression and poor prognosis. Even with the advent of targeted therapies and a multimodal approach, IBC is often treatment refractory and a therapeutic challenge for all subtypes, including ER+ and HER-2 amplified (HER2+) disease (Masuda et al). Therefore identifying mechanisms of resistance to molecularly targeted therapy could provide clues to improve management and outcome. Recent studies comparing the gene expression profiles of IBC tumors with non-IBC demonstrated that HER2+ IBC have increased mTOR signaling compared to their non-IBC counterparts (Iwamoto et al). mTOR activation is a mechanism for Trastuzumab resistance and may contribute to treatment resistance in HER2+ IBC. The availability of molecular diagnostics evaluating phosphoProteins is an appealing approach to predict treatment-sensitivity and select more effective combinations. Methods This is an observational analysis of 12 IBC patients who had tissue biopsy after progression on standard therapies including HER-2 targeted therapies. Tissue analysis for expression of cancer-related phosphoProteins was performed using TheraLink™. The TheraLink™ assay uses reverse-phase Protein Microarrays (RPMA) to quantify HER1, HER2, and HER3 receptor overexpression; it also evaluates for phosphorylation of the receptor which indicates activation. Phosphorylation of HER downstream signaling pathways such as JAK2, AKT/mTOR and MEK1/2 are also detected. Additionally, next generation sequencing (NGS) using FoundationOne™ was performed if sufficient tissue was available. Results All patients had IBC and most had metastatic disease (83%). According to subtype 25% of patients were ER+/HER2-, 42% ER+/HER2+, 25% ER-/HER2+, 8% ER-/HER2-. 58% of tumors demonstrated HER1 activation, 75% had HER2 activation and 58% had HER3 activation. Interestingly, 83% had mTOR activation, and most of these patients also had accumulation of its downstream Proteins, S6 ribosomal Protein and 4E-BP-1. 78% of patients with HER2 activation also had mTOR activation. Two of the 4 patients who were HER2- by IHC/FISH had HER2 activation by RPMA. Six patients also had NGS on tissue; 75% had concordance between HER2 activation on TheraLink™ and ERBB2 amplification on NGS, 67% had concordance with mTOR activation on TheraLink™ and mutation in the mTOR pathway (PIK3CA mutation or PTEN loss) on NGS. One patient with triple negative, chemo-refractory IBC who underwent 3 lines of neoadjuvant therapy prior to bilateral mastectomy was found to have HER1, HER2, HER3 and mTOR activation; she was started on lapatinib and capecitabine and remains with no recurrent disease and on treatment. Conclusions Patients with IBC often have activation of members of the HER family and mTOR pathway indicating molecular targets and potential mechanisms of resistance in IBC. The concomitant use of NGS and RPMA is an intriguing approach to molecular diagnostics in this aggressive and treatment refractory disease providing additional information on pathway activation leading to expanded therapeutic options. Future prospective studies should clarify the potential impact in treatment selection and outcome. Citation Format: Laura Austin, Kimberly Limentani, Juan Palazzo, Tiffany Avery, Rebecca Jaslow, Ron Hencin, Emanuel F Petricoin, Massimo Cristofanilli. Identifying molecular targets and mechanisms of treatment resistance in inflammatory breast cancer (IBC) using reverse-phase Protein Microarrays (RPMA) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-04-02.
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reverse phase Protein Microarrays application to biomarker discovery and translational medicine
Expert Review of Molecular Diagnostics, 2007Co-Authors: Amy Vanmeter, Virginia Espina, Lance A Liotta, Michele Signore, Mariaelena Pierobon, Emanuel F PetricoinAbstract:Mapping of Protein signaling networks within tumors can identify new targets for therapy and provide a means to stratify patients for individualized therapy. Kinases are important drug targets, as such kinase network information could become the basis for development of therapeutic strategies for improving treatment outcome. An urgent clinical goal is to identify functionally important molecular networks associated with subpopulations of patients that may not respond to conventional combination chemotherapy. Reverse-phase Protein Microarrays are a technology platform designed for quantitative, multiplexed analysis of specific phosphorylated, cleaved, or total (phosphorylated and nonphosphorylated) forms of cellular Proteins from a limited amount of sample. This class of microarray can be used to interrogate cellular samples, serum or body fluids. This review focuses on the application of reverse-phase Protein Microarrays for translational research and therapeutic drug target discovery.
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physicochemically modified silicon as a substrate for Protein Microarrays
Biomaterials, 2007Co-Authors: Jasper A Nijdam, Virginia Espina, Emanuel F Petricoin, David Geho, Mark Mingcheng Cheng, Roberta Fedele, Paul C Herrmann, Keith Killian, Lance A LiottaAbstract:Reverse phase Protein Microarrays (RPMA) enable high throughput screening of posttranslational modifications of important signaling Proteins within diseased cells. One limitation of Protein-based molecular profiling is the lack of a PCR-like intrinsic amplification system for Proteins. Enhancement of Protein microarray sensitivities is an important goal, especially because many molecular targets within patient tissues are of low abundance. The ideal array substrate will have a high Protein-binding affinity and low intrinsic signal. To date, nitrocellulose-coated glass has provided an effective substrate for Protein binding in the microarray format when using chromogenic detection systems. As fluorescent systems, such as quantum dots, are explored as potential reporter agents, the intrinsic fluorescent properties of nitrocellulose-coated glass slides limit the ability to image Microarrays for extended periods of time where increases in net sensitivity can be attained. Silicon, with low intrinsic autofluorescence, is being explored as a potential microarray surface. Native silicon has low binding potential. Through titrated reactive ion etching (RIE), varying surface areas have been created on silicon in order to enhance Protein binding. Further, via chemical modification, reactive groups have been added to the surfaces for comparison of relative Protein binding. Using this combinatorial method of surface roughening and surface coating, 3-aminopropyltriethoxysilane (APTES) and mercaptopropyltrimethoxysilane (MPTMS) treatments were shown to transform native silicon into a Protein-binding substrate comparable to nitrocellulose.
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pegylated steptavidin conjugated quantum dots are effective detection elements for reverse phase Protein Microarrays
Bioconjugate Chemistry, 2005Co-Authors: David Geho, Virginia Espina, Julia Wulfkuhle, Paul C Herrmann, Nicholas Lahar, Prem Gurnani, Michael L Huebschman, Alice Shi, Harold R Garner, Emanuel F PetricoinAbstract:Protein microarray technologies provide a means of investigating the proteomic content of clinical biopsy specimens in order to determine the relative activity of key nodes within cellular signaling pathways. A particular kind of Protein microarray, the reverse-phase microarray, is being evaluated in clinical trials because of its potential to utilize limited amounts of cellular material obtained through biopsy. Using this approach, cellular lysates are arrayed in dilution curves on nitrocellulose substrates for subsequent probing with antibodies. To improve the sensitivity and utility of reverse-phase Microarrays, we tested whether a new reporter technology as well as a new detection instrument could enhance microarray performance. We describe the use of an inorganic fluorescent nanoparticle conjugated to streptavidin, Qdot 655 Sav, in a reverse-phase Protein microarray format for signal pathway profiling. Moreover, a pegylated form of this bioconjugate, Qdot 655 Sav, is found to have superior detection characteristics in assays performed on cellular Protein extracts over the nonpegylated form of the bioconjugate. Hyperspectral imaging of the quantum dot microarray enabled unamplified detection of signaling Proteins within defined cellular lysates, which indicates that this approach may be amenable to multiplexed, high-throughput reverse-phase Protein Microarrays in which numerous analytes are measured in parallel within a single spot.
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Protein microarray detection strategies focus on direct detection technologies
Journal of Immunological Methods, 2004Co-Authors: Virginia Espina, Elisa C Woodhouse, Julia Wulfkuhle, Heather D Asmussen, Emanuel F Petricoin, Lance A LiottaAbstract:Protein Microarrays are being utilized for functional proteomic analysis, providing information not obtainable by gene arrays. Microarray technology is applicable for studying Protein-Protein, Protein-ligand, kinase activity and posttranslational modifications of Proteins. A precise and sensitive Protein microarray, the direct detection or reverse-phase microarray, has been applied to ongoing clinical trials at the National Cancer Institute for studying phosphorylation events in EGF-receptor-mediated cell signaling pathways. The variety of microarray applications allows for multiple, creative microarray designs and detection strategies. Herein, we discuss detection strategies and challenges for Protein microarray technology, focusing on direct detection of Protein Microarrays.
Zhuo Chen - One of the best experts on this subject based on the ideXlab platform.
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plasmonic substrates for multiplexed Protein Microarrays with femtomolar sensitivity and broad dynamic range
Nature Communications, 2011Co-Authors: Scott M Tabakman, Lana Lau, Joshua T Robinson, Jordan V Price, Sarah Sherlock, Hailiang Wang, Bo Zhang, Zhuo Chen, Stephanie Tangsombatvisit, Justin A JarrellAbstract:Protein chips are widely used for high-throughput proteomic analysis, but to date, the low sensitivity and narrow dynamic range have limited their capabilities in diagnostics and proteomics. Here we present Protein Microarrays on a novel nanostructured, plasmonic gold film with near-infrared fluorescence enhancement of up to 100-fold, extending the dynamic range of Protein detection by three orders of magnitude towards the fM regime. We employ plasmonic Protein Microarrays for the early detection of a cancer biomarker, carcinoembryonic antigen, in the sera of mice bearing a xenograft tumour model. Further, we demonstrate a multiplexed autoantigen array for human autoantibodies implicated in a range of autoimmune diseases with superior signal-to-noise ratios and broader dynamic range compared with commercial nitrocellulose and glass substrates. The high sensitivity, broad dynamic range and easy adaptability of plasmonic Protein chips presents new opportunities in proteomic research and diagnostics applications. Protein Microarrays are useful both in basic research and also in disease monitoring and diagnosis, but their dynamic range is limited. By using plasmonic gold substrates with near-infrared fluorescent enhancement, Tabakman et al. demonstrate a multiplexed Protein array with improved detection limits and dynamic range.
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plasmonic substrates for multiplexed Protein Microarrays with femtomolar sensitivity and broad dynamic range
Nature Communications, 2011Co-Authors: Scott M Tabakman, Lana Lau, Joshua T Robinson, Jordan V Price, Sarah Sherlock, Hailiang Wang, Bo Zhang, Zhuo Chen, Stephanie Tangsombatvisit, Justin A JarrellAbstract:Protein chips are widely used for high-throughput proteomic analysis, but to date, the low sensitivity and narrow dynamic range have limited their capabilities in diagnostics and proteomics. Here we present Protein Microarrays on a novel nanostructured, plasmonic gold film with near-infrared fluorescence enhancement of up to 100-fold, extending the dynamic range of Protein detection by three orders of magnitude towards the fM regime. We employ plasmonic Protein Microarrays for the early detection of a cancer biomarker, carcinoembryonic antigen, in the sera of mice bearing a xenograft tumour model. Further, we demonstrate a multiplexed autoantigen array for human autoantibodies implicated in a range of autoimmune diseases with superior signal-to-noise ratios and broader dynamic range compared with commercial nitrocellulose and glass substrates. The high sensitivity, broad dynamic range and easy adaptability of plasmonic Protein chips presents new opportunities in proteomic research and diagnostics applications.
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Protein Microarrays with carbon nanotubes as multicolor raman labels
Nature Biotechnology, 2008Co-Authors: Zhuo Chen, Scott M Tabakman, Andrew P Goodwin, Michael G Kattah, Dan Daranciang, Xinran Wang, Guangyu Zhang, Zhuang Liu, Paul J Utz, Kaili JiangAbstract:The picomolar sensitivity of fluorescence-based Protein detection limits the use of Protein arrays in research and clinical diagnosis. Chen et al. use antibody-tagged single-walled carbon nanotubes as multicolor Raman labels to detect femtomolar levels of serum analytes over a wide dynamic range.
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Protein Microarrays with carbon nanotubes as multicolor raman labels
Nature Biotechnology, 2008Co-Authors: Zhuo Chen, Scott M Tabakman, Andrew P Goodwin, Michael G Kattah, Dan Daranciang, Xinran Wang, Guangyu Zhang, Zhuang Liu, Paul J Utz, Kaili JiangAbstract:The current sensitivity of standard fluorescence-based Protein detection limits the use of Protein arrays in research and clinical diagnosis. Here, we use functionalized, macromolecular single-walled carbon nanotubes (SWNTs) as multicolor Raman labels for highly sensitive, multiplexed Protein detection in an arrayed format. Unlike fluorescence methods, Raman detection benefits from the sharp scattering peaks of SWNTs with minimal background interference, affording a high signal-to-noise ratio needed for ultra-sensitive detection. When combined with surface-enhanced Raman scattering substrates, the strong Raman intensity of SWNT tags affords Protein detection sensitivity in sandwich assays down to 1 fM--a three-order-of-magnitude improvement over most reports of fluorescence-based detection. We use SWNT Raman tags to detect human autoantibodies against Proteinase 3, a biomarker for the autoimmune disease Wegener's granulomatosis, diluted up to 10(7)-fold in 1% human serum. SWNT Raman tags are not subject to photobleaching or quenching. By conjugating different antibodies to pure (12)C and (13)C SWNT isotopes, we demonstrate multiplexed two-color SWNT Raman-based Protein detection.
