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Amy E Herr - One of the best experts on this subject based on the ideXlab platform.
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Subcellular Western Blotting of single cells.
Microsystems & nanoengineering, 2017Co-Authors: Kevin A Yamauchi, Amy E HerrAbstract:Although immunoassays are the de facto standard for determining subcellular protein localization in individual cells, antibody probe cross-reactivity and fixation artifacts remain confounding factors. To enhance selectivity while providing single-cell resolution, we introduce a subcellular Western Blotting technique capable of separately assaying proteins in the 14 pL cytoplasm and 2 pL nucleus of individual cells. To confer precision fluidic control, we describe a passive multilayer microdevice that leverages the rapid transport times afforded by miniaturization. After isolating single cells in microwells, we apply single-cell differential detergent fractionation to lyse and Western blot the cytoplasmic lysate, whereas the nucleus remains intact in the microwell. Subsequently, we lyse the intact nucleus and Western blot the nuclear lysate. To index each protein analysis to the originating subcellular compartment, we utilize bi-directional electrophoresis, a multidimensional separation that assays the lysate from each compartment in a distinct region of the separation axis. Single-cell bi-directional electrophoresis eliminates the need for semi-subjective image segmentation algorithms required in immunocytochemistry. The subcellular, single-cell Western blot is demonstrated for six targets per cell, and successfully localizes spliceosome-associated proteins solubilized from large protein and RNA complexes, even for closely sized proteins (a 7 kDa difference). Measurement of NF-κB translocation dynamics in unfixed cells at 15-min intervals demonstrates reduced technical variance compared with immunofluorescence. This chemical cytometry assay directly measures the nucleocytoplasmic protein distribution in individual unfixed cells, thus providing insight into protein signaling in heterogeneous cell populations.
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single cell resolution Western Blotting
Nature Protocols, 2016Co-Authors: Chichih Kang, Kevin A Yamauchi, Julea Vlassakis, Elly Sinkala, Todd A Duncombe, Amy E HerrAbstract:This protocol describes how to perform Western Blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional Western Blotting, single-cell Western Blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a Blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4-6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine.
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single cell Western Blotting
Nature Methods, 2014Co-Authors: Alex J Hughes, Chichih Kang, Dawn P Spelke, Zhuchen Xu, David V Schaffer, Amy E HerrAbstract:To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct ∼10(3) concurrent single-cell Western blots (scWesterns) in ∼4 h. A microscope slide supporting a 30-μm-thick photoactive polyacrylamide gel enables Western Blotting: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated Blotting to immobilize proteins and antibody probing. We applied this scWestern method to monitor single-cell differentiation of rat neural stem cells and responses to mitogen stimulation. The scWestern quantified target proteins even with off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supported analyses of low starting cell numbers (∼200) when integrated with FACS. The scWestern overcomes limitations of antibody fidelity and sensitivity in other single-cell protein analysis methods and constitutes a versatile tool for the study of complex cell populations at single-cell resolution.
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microfluidic integration of Western Blotting is enabled by electrotransfer assisted sodium dodecyl sulfate dilution
Analyst, 2013Co-Authors: Amy E HerrAbstract:We integrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent antibody probing in a single, monolithic microdevice to realize microfluidic Western Blotting. A hurdle to successful on-chip Western Blotting lies in restoring antibody recognition of previously sized (denatured, reduced) proteins. To surmount this hurdle, we locally dilute free SDS from SDS–protein complexes using differential electromigration of the species during electrotransfer between SDS-PAGE and Blotting regions of a microchamber. Local dilution of SDS minimizes re-association of SDS with proteins offering means to restore antibody binding affinity to proteins after SDS-PAGE. To achieve automated, programmable operation in a single instrument, we utilize a 1 × 2 mm2 glass microchamber photopatterned with spatially distinct, contiguous polyacrylamide regions for SDS-PAGE, electrotransfer, and antibody Blotting. Optimization of both the SDS-PAGE and electrotransfer conditions yields transfer distances of <1 mm (40 s). The Western blot is completed in 180 s, with fully automated assay operation using programmable voltage control. After SDS-PAGE and electrotransfer, we observe ∼80% capture of protein band mass on the Blotting region for a model protein, C-reactive protein. This novel microfluidic Western blot approach introduces fine transport control for in-transit protein handling to form the basis for an automated, rapid alternative to conventional slab-gel Western Blotting.
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microfluidic Western Blotting
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Alex J Hughes, Amy E HerrAbstract:Rapid, quantitative Western Blotting is a long-sought bioanalytical goal in the life sciences. To this end, we describe a Western Blotting assay conducted in a single glass microchannel under purely electronic control. The μWestern blot is comprised of multiple steps: sample enrichment, protein sizing, protein immobilization (Blotting), and in situ antibody probing. To validate the microfluidic assay, we apply the μWestern blot to analyses of human sera (HIV immunoreactivity) and cell lysate (NFκB). Analytical performance advances are achieved, including: short durations of 10–60 min, multiplexed analyte detection, mass sensitivity at the femtogram level, high-sensitivity 50-pM detection limits, and quantitation capability over a 3.6-log dynamic range. Performance gains are attributed to favorable transport and reaction conditions on the microscale. The multistep assay design relies on a photopatternable (blue light) and photoreactive (UV light) polyacrylamide gel. This hydrophilic polymer constitutes both a separation matrix for protein sizing and, after brief UV exposure, a protein immobilization scaffold for subsequent antibody probing of immobilized protein bands. We observe protein capture efficiencies exceeding 75% under sizing conditions. This compact microfluidic design supports demonstration of a 48-plex μWestern blot in a standard microscope slide form factor. Taken together, the μWestern blot establishes a foundation for rapid, targeted proteomics by merging exceptional specificity with the throughput advantages of multiplexing, as is relevant to a broad range of biological inquiry.
Edita Aksamitiene - One of the best experts on this subject based on the ideXlab platform.
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multistrip Western Blotting a tool for comparative quantitative analysis of multiple proteins
Methods of Molecular Biology, 2015Co-Authors: Edita Aksamitiene, Jan B Hoek, Anatoly KiyatkinAbstract:: The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical Western Blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip Western Blotting is a modified immunoBlotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip Western Blotting increases data output per single Blotting cycle up to tenfold; allows concurrent measurement of up to nine different total and/or posttranslationally modified protein expression obtained from the same loading of the sample; and substantially improves the data accuracy by reducing immunoBlotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data and therefore is advantageous to apply in biomedical diagnostics, systems biology, and cell signaling research.
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Multistrip Western Blotting to increase quantitative data output.
Methods of Molecular Biology, 2009Co-Authors: Anatoly Kiyatkin, Edita AksamitieneAbstract:The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical Western Blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip Western Blotting is a modified immunoBlotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip Western Blotting increases the data output per single Blotting cycle up to ten-fold, allows concurrent monitoring of up to nine different proteins from the same loading of the sample, and substantially improves the data accuracy by reducing immunoBlotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data, and therefore is beneficial to apply in biomedical diagnostics, systems biology and cell signaling research.
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multistrip Western Blotting to increase quantitative data output
Electrophoresis, 2007Co-Authors: Jan B Hoek, Edita Aksamitiene, Boris N Kholodenko, Anatoly KiyatkinAbstract:The qualitative and quantitative measurements of protein abundance and protein modification states are essential in understanding their role in diverse cellular processes. Traditional Western Blotting technique, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. We propose a modified immunoBlotting procedure, which is based on simultaneous transfer of proteins from multiple gel-strips onto the same membrane, and is compatible with any conventional gel electrophoresis system. As a result, the data output per single Blotting cycle can readily be increased up to ten-fold. In contrast to the traditional "one protein detection per electrophoresis cycle", this procedure allows simultaneous monitoring of up to nine different proteins. In addition to maintaining the ability to detect picogram quantities of protein, the modified system substantially improves data accuracy by reducing signal errors by two-fold. Multistrip Western Blotting procedure allows making statistically reliable side-by-side comparisons of different or repeated sets of data. Compared to the traditional methods, this approach provides a more economical, reproducible, and effective procedure, facilitating the generation of large amounts of high-quality quantifiable data.
Anatoly Kiyatkin - One of the best experts on this subject based on the ideXlab platform.
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multistrip Western Blotting a tool for comparative quantitative analysis of multiple proteins
Methods of Molecular Biology, 2015Co-Authors: Edita Aksamitiene, Jan B Hoek, Anatoly KiyatkinAbstract:: The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical Western Blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip Western Blotting is a modified immunoBlotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip Western Blotting increases data output per single Blotting cycle up to tenfold; allows concurrent measurement of up to nine different total and/or posttranslationally modified protein expression obtained from the same loading of the sample; and substantially improves the data accuracy by reducing immunoBlotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data and therefore is advantageous to apply in biomedical diagnostics, systems biology, and cell signaling research.
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Multistrip Western Blotting to increase quantitative data output.
Methods of Molecular Biology, 2009Co-Authors: Anatoly Kiyatkin, Edita AksamitieneAbstract:The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical Western Blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip Western Blotting is a modified immunoBlotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip Western Blotting increases the data output per single Blotting cycle up to ten-fold, allows concurrent monitoring of up to nine different proteins from the same loading of the sample, and substantially improves the data accuracy by reducing immunoBlotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data, and therefore is beneficial to apply in biomedical diagnostics, systems biology and cell signaling research.
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multistrip Western Blotting to increase quantitative data output
Electrophoresis, 2007Co-Authors: Jan B Hoek, Edita Aksamitiene, Boris N Kholodenko, Anatoly KiyatkinAbstract:The qualitative and quantitative measurements of protein abundance and protein modification states are essential in understanding their role in diverse cellular processes. Traditional Western Blotting technique, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. We propose a modified immunoBlotting procedure, which is based on simultaneous transfer of proteins from multiple gel-strips onto the same membrane, and is compatible with any conventional gel electrophoresis system. As a result, the data output per single Blotting cycle can readily be increased up to ten-fold. In contrast to the traditional "one protein detection per electrophoresis cycle", this procedure allows simultaneous monitoring of up to nine different proteins. In addition to maintaining the ability to detect picogram quantities of protein, the modified system substantially improves data accuracy by reducing signal errors by two-fold. Multistrip Western Blotting procedure allows making statistically reliable side-by-side comparisons of different or repeated sets of data. Compared to the traditional methods, this approach provides a more economical, reproducible, and effective procedure, facilitating the generation of large amounts of high-quality quantifiable data.
Chichih Kang - One of the best experts on this subject based on the ideXlab platform.
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single cell resolution Western Blotting
Nature Protocols, 2016Co-Authors: Chichih Kang, Kevin A Yamauchi, Julea Vlassakis, Elly Sinkala, Todd A Duncombe, Amy E HerrAbstract:This protocol describes how to perform Western Blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional Western Blotting, single-cell Western Blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a Blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4-6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine.
-
single cell Western Blotting
Nature Methods, 2014Co-Authors: Alex J Hughes, Chichih Kang, Dawn P Spelke, Zhuchen Xu, David V Schaffer, Amy E HerrAbstract:To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct ∼10(3) concurrent single-cell Western blots (scWesterns) in ∼4 h. A microscope slide supporting a 30-μm-thick photoactive polyacrylamide gel enables Western Blotting: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated Blotting to immobilize proteins and antibody probing. We applied this scWestern method to monitor single-cell differentiation of rat neural stem cells and responses to mitogen stimulation. The scWestern quantified target proteins even with off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supported analyses of low starting cell numbers (∼200) when integrated with FACS. The scWestern overcomes limitations of antibody fidelity and sensitivity in other single-cell protein analysis methods and constitutes a versatile tool for the study of complex cell populations at single-cell resolution.
Alex J Hughes - One of the best experts on this subject based on the ideXlab platform.
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single cell Western Blotting
Nature Methods, 2014Co-Authors: Alex J Hughes, Chichih Kang, Dawn P Spelke, Zhuchen Xu, David V Schaffer, Amy E HerrAbstract:To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct ∼10(3) concurrent single-cell Western blots (scWesterns) in ∼4 h. A microscope slide supporting a 30-μm-thick photoactive polyacrylamide gel enables Western Blotting: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated Blotting to immobilize proteins and antibody probing. We applied this scWestern method to monitor single-cell differentiation of rat neural stem cells and responses to mitogen stimulation. The scWestern quantified target proteins even with off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supported analyses of low starting cell numbers (∼200) when integrated with FACS. The scWestern overcomes limitations of antibody fidelity and sensitivity in other single-cell protein analysis methods and constitutes a versatile tool for the study of complex cell populations at single-cell resolution.
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microfluidic Western Blotting
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Alex J Hughes, Amy E HerrAbstract:Rapid, quantitative Western Blotting is a long-sought bioanalytical goal in the life sciences. To this end, we describe a Western Blotting assay conducted in a single glass microchannel under purely electronic control. The μWestern blot is comprised of multiple steps: sample enrichment, protein sizing, protein immobilization (Blotting), and in situ antibody probing. To validate the microfluidic assay, we apply the μWestern blot to analyses of human sera (HIV immunoreactivity) and cell lysate (NFκB). Analytical performance advances are achieved, including: short durations of 10–60 min, multiplexed analyte detection, mass sensitivity at the femtogram level, high-sensitivity 50-pM detection limits, and quantitation capability over a 3.6-log dynamic range. Performance gains are attributed to favorable transport and reaction conditions on the microscale. The multistep assay design relies on a photopatternable (blue light) and photoreactive (UV light) polyacrylamide gel. This hydrophilic polymer constitutes both a separation matrix for protein sizing and, after brief UV exposure, a protein immobilization scaffold for subsequent antibody probing of immobilized protein bands. We observe protein capture efficiencies exceeding 75% under sizing conditions. This compact microfluidic design supports demonstration of a 48-plex μWestern blot in a standard microscope slide form factor. Taken together, the μWestern blot establishes a foundation for rapid, targeted proteomics by merging exceptional specificity with the throughput advantages of multiplexing, as is relevant to a broad range of biological inquiry.
