A study of rubisco through western blotting and tissue printing techniques.
Ontology highlight
ABSTRACT:
SUBMITTER: Ma Z
PROVIDER: S-EPMC2689150 | biostudies-literature | 2009
REPOSITORIES: biostudies-literature
Publications
A study of rubisco through western blotting and tissue printing techniques.
Ma Zhong Z Cooper Cynthia C Kim Hyun-Joo HJ Janick-Buckner Diane D
CBE life sciences education 20090101 2
We describe a laboratory exercise developed for a cell biology course for second-year undergraduate biology majors. It was designed to introduce undergraduates to the basic molecular biology techniques of Western blotting and immunodetection coupled with the technique of tissue printing in detecting the presence, relative abundance, and distribution of ribulose-1,5-bisphosphate carboxylase in various plant materials. Pre- and postlab surveys indicated significant postlab gains in student underst ...[more]
Similar Datasets
Project description: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 ?Western 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 ?Western blot to analyses of human sera (HIV immunoreactivity) and cell lysate (NF?B). 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 ?Western blot in a standard microscope slide form factor. Taken together, the ?Western 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.
| S-EPMC3535594 | biostudies-literature
Project description: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.
| S-EPMC4077215 | biostudies-literature
Project description:Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.
| S-EPMC4545415 | biostudies-literature
Project description: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.
| S-EPMC5511750 | biostudies-literature
Project description: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.
| S-EPMC5764185 | biostudies-literature
Project description:Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps.
| S-EPMC5113996 | biostudies-literature
Project description:We describe a microfluidic Western blot assay (?Western) using a Tris tricine discontinuous buffer system suitable for analyses of a wide molecular mass range (6.5-116 kDa). The Tris tricine ?Western is completed in an enclosed, straight glass microfluidic channel housing a photopatterned polyacrylamide gel that incorporates a photoactive benzophenone methacrylamide monomer. Upon brief ultraviolet (UV) light exposure, the hydrogel toggles from molecular sieving for size-based separation to a covalent immobilization scaffold for in situ antibody probing. Electrophoresis controls all assay stages, affording purely electronic operation with no pumps or valves needed for fluid control. Electrophoretic introduction of antibody into and along the molecular sieving gel requires that the probe must traverse through (i) a discontinuous gel interface central to the transient isotachophoresis used to achieve high-performance separations and (ii) the full axial length of the separation gel. In-channel antibody probing of small molecular mass species is especially challenging, since the gel must effectively sieve small proteins while permitting effective probing with large-molecular-mass antibodies. To create a well-controlled gel interface, we introduce a fabrication method that relies on a hydrostatic pressure mismatch between the buffer and polymer precursor solution to eliminate the interfacial pore-size control issues that arise when a polymerizing polymer abuts a nonpolymerizing polymer solution. Combined with a new swept antibody probe plug delivery scheme, the Tris tricine ?Western blot enables 40% higher separation resolution as compared to a Tris glycine system, destacking of proteins down to 6.5 kDa, and a 100-fold better signal-to-noise ratio (SNR) for small pore gels, expanding the range of applicable biological targets.
| S-EPMC4222625 | biostudies-literature
Project description: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.
| S-EPMC2410211 | biostudies-literature
Project description:Traditional Western blotting is one of the most used analytical techniques in biological research. However, it can be time-consuming and suffer from a lack of reproducibility. Consequently, devices with different degrees of automation have been developed. These include semi-automated techniques and fully automated devices that replicate all stages downstream of the sample preparation, including sample size separation, immunoblotting, imaging, and analysis. We directly compared traditional Western blotting with two different automated systems, iBind™ Flex, which is a semi-automated system designed to perform the immunoblotting, and JESS Simple Western™, a fully automated and capillary-based system performing all steps downstream of sample preparation and loading, including imaging and image analysis. We found that a fully automated system can save time and importantly offer valuable sensitivity. This is particularly beneficial for limited sample amounts. The downside of automation is the cost of devices and reagents. Nevertheless, automation can be a good option to increase output and facilitate sensitive protein analyses.
| S-EPMC10142486 | biostudies-literature
Project description:Direct measurement of proteins from single cells has been realized at the microscale using microfluidic channels, capillaries, and semi-enclosed microwell arrays. Although powerful, these formats are constrained, with the enclosed geometries proving cumbersome for multistage assays, including electrophoresis followed by immunoprobing. We introduce a hybrid microfluidic format that toggles between a planar microwell array and a suspension of microparticles. The planar array is stippled in a thin sheet of polyacrylamide gel, for efficient single-cell isolation and protein electrophoresis of hundreds-to-thousands of cells. Upon mechanical release, array elements become a suspension of separation-encoded microparticles for more efficient immunoprobing due to enhanced mass transfer. Dehydrating microparticles offer improved analytical sensitivity owing to in-gel concentration of fluorescence signal for high-throughput single-cell targeted proteomics.
| S-EPMC7029799 | biostudies-literature