achieved remarkable miniaturization and scale-up of the Western blot workflow through a novel combination of highly multiplexed fluid handling and a large format slab gel ( 14). In an approach using slab gels, Ciaccio et al. Recent innovation in protein analysis tools recognizes the crucial protein separation stage and seeks to retain separation-based information. Because of the power of the assay and in light of these deficiencies, Western blotting requires continued innovation to improve throughput, minimize resource use, and advance analytical sensitivity and dynamic range ( 7). A single 12-lane Western blot requires ∼300 mL of buffer and, most importantly, 1 μg of each detection antibody per analyte of interest. Moreover, material and reagent consumption is extensive. In fact, antibody probing often requires an overnight incubation period to compensate for diffusional limitations on antibody equilibration with antigen captured on the blotting material. Several fundamental limitations impede performance, including slow mass transport.
Loading control western blot series#
The blotting membrane immobilizes the protein separation and is then subjected to a series of handling steps, including blocking of nonspecific membrane interactions through coating with a dummy protein (e.g., BSA), incubation with antibodies to accomplish the probing step, and washing before obtaining assay readout.
To prepare for probing, the sized proteins are transferred from the sieving gel to a blotting membrane.
Loading control western blot manual#
Although conceptually straightforward, substantial preparation and manual intervention are required. Target identity is then established by linking immunoaffinity information to molecular mass. Once protein size is determined, the protein separation is incubated with antibodies (probing), thus allowing detection of interactions. During PAGE, proteins electromigrate through a polyacrylamide sieving gel, allowing determination of molecular mass. In the first stage of Western blotting, protein sizing, samples are analyzed by denaturing PAGE. ( C) Modular interfacing of standard microscope slide-sized chips with a scalable electrode array accommodating 48 blots per chip in triplicate (144 microchannels). Finally, excess probe is electrophoretically driven out of each device and peak intensities determined by fluorescence micrograph analysis. The microfluidic workflow is comprised of: ( i) analyte stacking and SDS-PAGE within the PACTgel matrix ( ii) band capture (“blotting”) onto the benzophenone-decorated PACTgel in response to UV light (as opposed to transfer to a separate sheet of hydrophobic material in conventional Western blotting) ( iii) removal of SDS by brief electrophoretic washing and electrophoretic introduction of fluorescently labeled primary and (optionally) secondary detection antibodies specific to the target. Aspects of scale, reagent use, blotting efficiency, and probe-binding kinetics are illustrated by comparative schematics for the conventional ( A) and μWestern ( B) assays (δ indicates a diffusion boundary layer thickness). Single-microchannel μWestern assay design enables high device density formats. 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.įig. This compact microfluidic design supports demonstration of a 48-plex μWestern blot in a standard microscope slide form factor. We observe protein capture efficiencies exceeding 75% under sizing conditions. 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. The multistep assay design relies on a photopatternable (blue light) and photoreactive (UV light) polyacrylamide gel. Performance gains are attributed to favorable transport and reaction conditions on the microscale. 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. To validate the microfluidic assay, we apply the μWestern blot to analyses of human sera (HIV immunoreactivity) and cell lysate (NFκB). The μWestern blot is comprised of multiple steps: sample enrichment, protein sizing, protein immobilization (blotting), and in situ antibody probing. To this end, we describe a Western blotting assay conducted in a single glass microchannel under purely electronic control. Rapid, quantitative Western blotting is a long-sought bioanalytical goal in the life sciences.
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