The Experts below are selected from a list of 98142 Experts worldwide ranked by ideXlab platform
Stephen M Hewitt - One of the best experts on this subject based on the ideXlab platform.
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transfer and multiplex Immunoblotting of a paraffin embedded tissue
Methods of Molecular Biology, 2009Co-Authors: Joonyong Chung, Stephen M HewittAbstract:In the functional proteome era, the proteomic profiling of clinicopathologic annotated tissues is an essential step for mining and evaluations of candidate biomarkers for disease. Previously, application of routine proteomic methodologies to clinical tissue specimens has provided unsatisfactory results. Multiplex tissue Immunoblotting is a method of transferring proteins from a formalin-fixed, paraffin-embedded tissue section to a stack of membranes which can be applied to a conventional Immunoblotting method. A single tissue section can be transferred to up to ten membranes, each of which is probed with antibodies and detected with fluorescent tags. By this approach, total protein and target signals can be simultaneously determined on each membrane; hence each antibody is internally normalized. Phosphorylation-specific antibodies as well as antibodies that do not readily work well with paraffin-embedded tissue are applicable to the membranes, expanding the menu of antibodies that can be utilized with formalin-fixed tissue. This novel platform can provide quantitative detection retaining histomorphologic detail in clinical samples and has great potential to facilitate discovery and development of new diagnostic assays and therapeutic agents.
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transfer and multiplex Immunoblotting of a paraffin embedded tissue
Proteomics, 2006Co-Authors: Joonyong Chung, Till Braunschweig, Galina Baibakov, Mike Galperin, Arun Ramesh, Marek Skacel, Gallya Gannot, Vladimir Knezevic, Stephen M HewittAbstract:As we transition from genomics to the challenges of the functional proteome, new tools to explore the expression of proteins within tissue are essential. We have developed a method of transferring proteins from a formalin fixed, paraffin embedded tissues section to a stack of membranes which is then probed with antibodies for detection of individual epitopes. This method converts a traditional tissue section into a multiplex platform for expression profiling. A single tissue section can be transferred to up to ten membranes, each of which is probed with different antibodies, and detected with fluorescent secondary antibodies, and quantified by a microarray scanner. Total protein can be determined on each membrane, hence each antibody has its own normalization. This method works with phospho-specific antibodies as well as antibodies that do not readily work well with paraffin embedded tissue. This novel technique enables archival paraffin embedded tissue to be molecularly profiled in a rapid and quantifiable manner, and reduces the tissue microarray to a form of protein array. This method is a new tool for exploration of the vast archive of formalin fixed, paraffin embedded tissue, as well as a tool for translational medicine.
Amy E Herr - One of the best experts on this subject based on the ideXlab platform.
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multiplexed ion beam imaging readout of single cell Immunoblotting
bioRxiv, 2021Co-Authors: Gabriela Lomeli, Marc Bosse, Sean C Bendall, Michael Angelo, Amy E HerrAbstract:ABSTRACT Improvements in single-cell protein analysis are required to study the cell-to-cell variation inherent to diseases, including cancer. Single-cell Immunoblotting (scIB) offers proteoform detection specificity, but often relies on fluorescence-based readout and is therefore limited in multiplexing capability. Among rising multiplexed imaging methods is multiplexed ion beam imaging by time of flight (MIBI-TOF), a mass spectrometry imaging technology. MIBI-TOF employs metal-tagged antibodies that do not suffer from spectra overlap to the same degree as fluorophore-tagged antibodies. We report for the first-time MIBI-TOF of single-cell Immunoblotting (scIB-MIBI-TOF). The scIB assay subjects single-cell lysate to protein Immunoblotting on a microscale device consisting of a 50- to 75-μm thick hydrated polyacrylamide (PA) gel matrix for protein immobilization prior to in-gel immunoprobing. We confirm antibody-protein binding in the PA gel with indirect fluorescence readout of metal-tagged antibodies. Since MIBI-TOF is a layer-by-layer imaging technique, and our protein target is immobilized within a 3D PA gel layer, we characterize the protein distribution throughout the PA gel depth by fluorescence confocal microscopy and find that the highest signal-to-noise ratio is achieved by imaging the entirety of the PA gel depth. Accordingly, we report the required MIBI-TOF ion dose strength needed to image varying PA gel depths. Lastly, by imaging ~42% of PA gel depth with MIBI-TOF, we detect two isoelectrically separated TurboGFP (tGFP) proteoforms from individual glioblastoma cells, demonstrating that highly multiplexed mass spectrometry-based readout is compatible with scIB.
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3d projection electrophoresis for single cell Immunoblotting
bioRxiv, 2019Co-Authors: Samantha M Grist, Andoni P Mourdoukoutas, Amy E HerrAbstract:ABSTRACT While single-cell resolution immunoassays and mass spectrometry are powerful protein analysis tools, key analytical bottlenecks remain in target specificity, analytical sensitivity, and measurement throughput. Advances are needed to complement single-cell genomics and transcriptomics tools. Here, we introduce highly parallel single-cell immunoblots designed to detect protein targets directly in unfixed mammalian cells. The 3D microfluidic device is comprised of a photoactive polyacrylamide gel, having one face (x-y) patterned with a high-density microwell array for cell isolation and lysis. From each microwell, single-cell lysate is ‘electrophoretically projected’ into the 3rd dimension (z-axis) for protein electrophoresis, photo-capture, and immunoprobing. Design guidelines for electrophoresis are informed by numerical simulation and empirical analyses of 3D diffusion of protein lysate. Unlike serial interrogation with capillary sampling interfaces, no automation or precision alignment is required for concurrent analysis of hundreds of cells. Importantly, cell separations are nearly synchronous ( 2.5 cells/sec, which is 80X faster than serial sampling approaches, and perform 25 single-cell immunoblots per 1 mm2 of device area, a >10X increase over reported single-cell immunoblots. A straightforward device for parallel, synchronized single-cell Immunoblotting, projection electrophoresis should advance integration of direct measurement of protein expression into the emerging single-cell atlas of genomic and transcriptomic profiles.
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fabrication of an open microfluidic device for Immunoblotting
Analytical Chemistry, 2017Co-Authors: Philippe Abdelsayed, Kevin A Yamauchi, Rachel E Gerver, Amy E HerrAbstract:Given the wide adoption of polydimethylsiloxane (PDMS) for the rapid fabrication of microfluidic networks and the utility of polyacrylamide gel electrophoresis (PAGE), we develop a technique for fabrication of PAGE molecular sieving gels in PDMS microchannel networks. In developing the fabrication protocol, we trade-off constraints on materials properties of these two polymer materials: PDMS is permeable to O2 and the presence of O2 inhibits the polymerization of polyacrylamide. We present a fabrication method compatible with performing PAGE protein separations in a composite PDMS-glass microdevice, that toggles from an “enclosed” microchannel for PAGE and blotting to an “open” PA gel lane for immunoprobing and readout. To overcome the inhibitory effects of O2, we coat the PDMS channel with a 10% benzophenone solution, which quenches the inhibiting effect of O2 when exposed to UV, resulting in a PAGE-in-PDMS device. We then characterize the PAGE separation performance. Using a ladder of small-to-mid mass ...
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Automated microfluidic protein Immunoblotting
Nature Protocols, 2010Co-Authors: Amy E HerrAbstract:This protocol describes regional photopatterning of polyacrylamide gels in glass microfluidic devices as a platform for seamless integration of multiple assay steps. The technology enables rapid, automated protein Immunoblotting, demonstrated in this study for native western blotting. The fabrication procedure is straightforward and requires approximately 3 h from the start of gel photopatterning to completion of native protein western blotting, a substantial time savings over slab-gel Immunoblotting. The assay itself requires less than 5 min. Importantly, all assay stages are programmably controlled by a high-voltage power supply and monitored by an epifluorescence microscope equipped with a charge-coupled device camera. Our approach overcomes severe limitations associated with conventional Immunoblotting, including multiple steps requiring manual intervention, low throughput and substantial consumption of reagents. We also describe a simple chemical recycling protocol so that glass chips can be reused. The fabrication technique described forms the basis for a diverse suite of bioanalytical tools, including DNA/RNA blotting and multidimensional separations.
Stefaan Depraetere - One of the best experts on this subject based on the ideXlab platform.
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Shotgun proteomics, in-silico evaluation and Immunoblotting assays for allergenicity assessment of lesser mealworm, black soldier fly and their protein hydrolysates
Scientific Reports, 2020Co-Authors: Giulia Leni, Tullia Tedeschi, Andrea Faccini, Federico Pratesi, Claudia Folli, Ilaria Puxeddu, Paola Migliorini, Natasja Gianotten, Johan Jacobs, Stefaan DepraetereAbstract:Since 2018, insects have belonged the category of Novel Foods and the presence of allergens represents one of the main hazards connected to their consumption, also due to the potential cross-reactivity with Arthropoda pan-allergens. In the present work, the allergenicity assessment of black soldier fly and lesser mealworm was performed with a shotgun bottom-up proteomic approach combined with in-silico assessment, followed by IgG- and IgE-Immunoblotting experiments. The peptides identified, filtered for their abundance and robustness, belonged mainly to muscle proteins, which represented the most abundant protein group. The relevant potential allergens were in-silico identified by sequence similarity to known allergens, and among them tropomyosin resulted the most abundant insect allergen. IgG-Immunoblotting analysis with anti-Tropomyosin I antibodies and IgE-Immunoblotting assay with serum from patient allergic to crustacean tropomyosin were performed in order to assess the immunoreactivity in both insects. The immunoassays were carried out also on protein hydrolysates extracted by treating insects with Protease from Bacillus licheniformis (1%, 60 °C, pH 7.5). While IgG-Immunoblotting demonstrated the loss of immunoreactivity for both hydrolysates, IgE-Immunoblotting showed a partial immunoreactivity preservation, also after hydrolysis, in the case of black soldier fly hydrolysate, and a total loss of immunoreactivity for lesser mealworm hydrolysate
Takashi Hashimoto - One of the best experts on this subject based on the ideXlab platform.
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diagnosis of epidermolysis bullosa acquisita multicentre comparison of different assays for serum anti type vii collagen reactivity
Acta Dermato-venereologica, 2021Co-Authors: Maike M Holtsche, Takashi Hashimoto, Nina Van Beek, Giovanni Di Zenzo, Detlef Zillikens, C Prostsquarcioni, Matthias Titeux, Alain Hovnanian, Enno Schmidt, Stephanie GoletzAbstract:Epidermolysis bullosa acquisita is a pemphigoid disease characterized by autoantibodies against type VII collagen. This study compared the sensitivity and specificity of 6 diagnostic assays: type VII collagen non-collagenous domains enzyme-linked immunoassay (NC1/2 ELISA) (MBL, Nagoya, Japan); type VII collagen NC1 ELISA (Euroimmun, Lubeck, Germany); indirect immunofluorescence (IF) microscopy test based on the expression of recombinant NC1 in a human cell line (NC1 BIOCHIP®; Euroimmun); full-length recombinant type VII collagen ELISA; Immunoblotting with full-length type VII collagen in the extract of human dermis; and Immunoblotting with recombinant NC1. Immunoblotting with recombinant NC1 showed a sensitivity of 93.1% and specificity of 100%, followed by NC1 BIOCHIP® (sensitivity, 89.1%; specificity, 100%), Immunoblotting with human dermis (sensitivity, 87.1%; specificity 100%), NC1-ELISA (sensitivity 82.2%; specificity 98.6%), NC1/NC2 ELISA (sensitivity 88.1%; specificity 93.3%), and full-length type VII collagen ELISA (sensitivity 80.2%; specificity 93.8%).
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usefulness of Immunoblotting using purified laminin 5 in the diagnosis of anti laminin 5 cicatricial pemphigoid
Journal of Dermatological Science, 2003Co-Authors: Yoshiko Hisamatsu, Toshio Nishiyama, Satoshi Amano, Chihiro Matsui, Riza Ghohestani, Takashi HashimotoAbstract:Abstract Background: Anti-laminin 5 cicatricial pemphigoid (CP) is a mucosal-dominant subepithelial blistering disease characterized by IgG anti-basement membrane zone autoantibodies, that bind to dermal side of 1 M NaCl split skin and immunoprecipitate laminin 5. Laminin 5 is an epidermis-specific extracellular matrix consisting of α3, β3 and γ2 subunits. Recent studies have suggested that autoantibodies of anti-laminin 5 CP recognize the G domains of α3 subunit. Objective: We examined the reactivity of anti-laminin 5 CP by Immunoblotting using purified laminin 5 and recombinant proteins of α3 subunit. Method: We first examined the reactivity of anti-laminin 5 CP by Immunoblotting using purified laminin 5. To further investigate the epitopes in the G domains of α3 subunit, we produced recombinant proteins of G1–2, G1–3, G2–3, G3–5 domains, that covered entire G domain, and examined the reactivity of anti-laminin 5 CP sera with these recombinant proteins by Immunoblotting. Results: By Immunoblotting using purified laminin 5, 7 of 21 anti-laminin 5 CP sera reacted with α3 subunit, while 8 sera reacted with β3 subunit and one serum reacted with γ2 subunit. Two sera reacted with both α3 and β3 subunits, while seven sera did not show positive reactivity. This result indicates that the reactivity of anti-laminin 5 CP sera is much more heterogeneous, although the previous studies suggested that most sera reacted with α3 subunit. However, in the studies using recombinant proteins of G domains of α3 subunit, none of the CP sera, including the sera reactive with α3 subunit in purified laminin 5, reacted with any recombinant proteins. The reason for this negative reactivity with the recombinant proteins is not clear. Conclusion: The Immunoblotting using purified laminin 5 should be useful technique for the diagnosis of anti-laminin 5 CP, although the sensitivity was less than conventional immunoprecipitation analysis.
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Immunoblotting studies of linear IgA disease.
Journal of dermatological science, 1993Co-Authors: Marian Dmochowski, Takashi Hashimoto, Balbir S. Bhogal, Martin M. Black, John J. Zone, Takeji NishikawaAbstract:Patients with linear IgA deposits at the basement membrane zone (BMZ) detected by direct immunofluorescence (IF) may show diverse clinical and laboratory findings. The aim of this study was to investigate the issue of target antigens for linear IgA disease (LAD) antibodies. We examined sera from 46 adults and children with exclusive IgA deposits at the BMZ, by both indirect IF on 1 M NaCl split human skin and Immunoblotting. IgA anti-BMZ antibodies binding to the epidermal side of the split were found in 31 LAD sera. IgA anti-BMZ antibodies binding to the dermal side of the split were detected only in 4 LAD sera. No sera contained IgA anti-BMZ antibodies binding to both sides of the split. Immunoblotting revealed that 12 epidermal side-positive LAD sera reacted with the 97 kDa protein in the human epidermal extracts. Moreover, we found that 2 dermal side-positive LAD sera reacted with a protein of approximately 255 kDa on Immunoblotting of the dermal extract. We conclude that there are at least two types of LAD. However, the nature of target antigens for LAD antibodies remains to be determined.
Vicente Amato Neto - One of the best experts on this subject based on the ideXlab platform.
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Avaliação da resposta imune humoral frente a antígenos de Strongyloides venezuelensis
Revista da Sociedade Brasileira de Medicina Tropical, 2008Co-Authors: Carla Rodrigues Rigo, Susana Angelica Zevallos Lescano, Cláudia Regina De Marchi, Vicente Amato NetoAbstract:Strongyloidiasis affects 30 million people in 70 countries. This enteral parasitosis is usually diagnosed using parasitological tests based on hydrotropism or thermotropism of larvae eliminated in feces, but these tests have been shown to have low sensitivity. In this study, antigenic extracts were tested by means of ELISA, Immunoblotting and IFI, using filariform larvae of Strongyloides venezuelensis, a parasite of rodents that shows cross-reactions with Strongyloides stercoralis epitopes. Sensitivity of 89, 85 and 57% for the ELISA reaction and 100, 100 and 96% for Immunoblotting with the SAL, ZWIP and ZW antigens, and specificity of 90, 60 and 81% for ELISA and 96, 92 and 91% for Immunoblotting with the same antigens, were found in these assays.
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Avaliação da resposta imune humoral frente a antígenos de Strongyloides venezuelensis Evaluation of the humoral immune response to Strongyloides venezuelensis antigens
Sociedade Brasileira de Medicina Tropical (SBMT), 2008Co-Authors: Carla Rodrigues Rigo, Susana Angelica Zevallos Lescano, Cláudia Regina De Marchi, Vicente Amato NetoAbstract:A estrongiloidíase afeta 30 milhões de pessoas em 70 países. Usualmente, o diagnóstico dessa enteroparasitose é realizado por testes parasitológicos baseados no hidro termotropismo das larvas eliminadas nas fezes, porém esses têm se mostrado pouco sensíveis. Neste trabalho, extratos antigênicos foram testados pelas técnicas de ELISA, Immunoblotting e IFI, utilizando larvas filarióides de Strongyloides venezuelensis, parasita de roedores, que mostram reação cruzada com epítopos de Strongyloides stercoralis. Sensibilidade de 89, 85, 57% para a reação de ELISA e de 100, 100 e 96%, para o Immunoblotting com os antígenos SAL, ZWIP e ZW, e especificidade de 90, 60 e 81% para o ELISA e 96, 92 e 91% para o Immunoblotting para os mesmos antígenos, foram encontradas nestes ensaios.Strongyloidiasis affects 30 million people in 70 countries. This enteral parasitosis is usually diagnosed using parasitological tests based on hydrotropism or thermotropism of larvae eliminated in feces, but these tests have been shown to have low sensitivity. In this study, antigenic extracts were tested by means of ELISA, Immunoblotting and IFI, using filariform larvae of Strongyloides venezuelensis, a parasite of rodents that shows cross-reactions with Strongyloides stercoralis epitopes. Sensitivity of 89, 85 and 57% for the ELISA reaction and 100, 100 and 96% for Immunoblotting with the SAL, ZWIP and ZW antigens, and specificity of 90, 60 and 81% for ELISA and 96, 92 and 91% for Immunoblotting with the same antigens, were found in these assays
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avaliacao da resposta imune humoral frente a antigenos de strongyloides venezuelensis evaluation of the humoral immune response to strongyloides venezuelensis antigens
2008Co-Authors: Carla Rodrigues Rigo, Susana Angelica Zevallos Lescano, Cláudia Regina De Marchi, Vicente Amato NetoAbstract:Strongyloidiasis affects 30 million people in 70 countries. This enteral parasitosis is usually diagnosed using parasitological tests based on hydrotropism or thermotropism of larvae eliminated in feces, but these tests have been shown to have low sensitivity. In this study, antigenic extracts were tested by means of ELISA, Immunoblotting and IFI, using filariform larvae of Strongyloides venezuelensis, a parasite of rodents that shows cross-reactions with Strongyloides stercoralis epitopes. Sensitivity of 89, 85 and 57% for the ELISA reaction and 100, 100 and 96% for Immunoblotting with the SAL, ZWIP and ZW antigens, and specificity of 90, 60 and 81% for ELISA and 96, 92 and 91% for Immunoblotting with the same antigens, were found in these assays.
