Browsing by Subject "Western blot"

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    Application of pulse width modulation to a Western blotting device
    (2016) TruongVo, ThucNhi; Yokota, Hiroki
    One of the critical steps in a current Western blot technique is a blotting process, which in general requires one electrophoretic gel for every protein species to be analyzed. In most cases, multiple protein species are analyzed simultaneously and thus it is necessary for a scientist to run multiple gels. In order to make it possible to analyze multiple protein species from a single gel, a novel blotting device, BlotMan, was employed in this study. Designed by Dr. Chien’s group (YC Bioelectric), BlotMan uses pulse width modulation (PWM) for applying a protein size-dependent voltage during a blotting process. In this study, the differential average voltage profile, depending on protein size (e.g. 17 kDa to 140 kDa), was built and enabled BlotMan to transfer all protein species in equal efficiency regardless of the protein size. Furthermore, Blot- Man consists of a user-friendly, custom-made interface box, which can be remotely controlled by a smart phone. BlotMan’s capability was evaluated using standard protein markers, as well as protein samples that were isolated from chondrosarcoma cells (SW1353) and breast cancer cells (MDA-MB-213). The experimental results revealed that BlotMan was capable of generating 5 blotting membranes from a single gel simultaneously. Protein species such as c-Src, eukaryotic translation initiation factor 2 alpha (eIF2α) and its phosphorylated form (p-eIF2α), lamin B, and β-actin were successfully detected. It is also demonstrated that compared to a regular constant voltage, PWM signals improved transfer efficiency and a signal-to-noise ratio. In conclusion, this study demonstrated that BlotMan was able to facilitate Western blotting analysis by generating multiple blotting membranes from a single gel with an improved signal-to-noise ratio. Further analysis is recommended for understanding the mechanism of PWMts action on transfer efficiency and noise reduction.
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    Heregulin Activity Assays for Residual Testing of Cell Therapy Products
    (BMC, 2021-11-12) Monje, Paula V.; Bacallao, Ketty; Aparicio, Gabriela I.; Lalwani, Anil; Neurological Surgery, School of Medicine
    Background: Heregulin is a ligand for the protooncogene product ErbB/HER that acts as a key mitogenic factor for human Schwann cells (hSCs). Heregulin is required for sustained hSC growth in vitro but must be thoroughly removed before cell collection for transplantation due to potential safety concerns. The goal of this study was to develop simple cell-based assays to assess the effectiveness of heregulin addition to and removal from aliquots of hSC culture medium. These bioassays were based on the capacity of a β1-heregulin peptide to elicit ErbB/HER receptor signaling in adherent ErbB2+/ErbB3+ cells. Results: Western blotting was used to measure the activity of three different β1-heregulin/ErbB-activated kinases (ErbB3/HER3, ERK/MAPK and Akt/PKB) using phospho-specific antibodies against key activating residues. The duration, dose-dependency and specificity of β1-heregulin-initiated kinase phosphorylation were investigated, and controls were implemented for assay optimization and reproducibility to detect β1-heregulin activity in the nanomolar range. Results from these assays showed that the culture medium from transplantable hSCs elicited no detectable activation of the aforementioned kinases in independent rounds of testing, indicating that the implemented measures can ensure that the final hSC product is devoid of bioactive β1-heregulin molecules prior to transplantation. Conclusions: These assays may be valuable to detect impurities such as undefined soluble factors or factors for which other biochemical or biological assays are not yet available. Our workflow can be modified as necessary to determine the presence of ErbB/HER, ERK, and Akt activators other than β1-heregulin using native samples, such as fresh isolates from cell- or tissue extracts in addition to culture medium.
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    Low-Level Vagus Nerve Stimulation Upregulates Small Conductance Calcium Activated Potassium Channels in the Stellate Ganglion
    (Elsevier, 2013) Shen, Mark J.; Chang, Hao-Che; Park, Hyung-Wook; Akingba, A. George; Chang, Po-Cheng; Zhang, Zheng; Lin, Shien-Fong; Shen, Changyu; Chen, Lan S.; Chen, Zhenhui; Fishbein, Michael C.; Chiamvimonvat, Nipavan; Chen, Peng-Sheng; Medicine, School of Medicine
    Background: Small conductance calcium-activated potassium (SK) channels are responsible for afterhyperpolarization that suppresses nerve discharges. Objectives: To test the hypothesis that low-level vagus nerve stimulation (LL-VNS) leads to the upregulation of SK2 proteins in the left stellate ganglion. Methods: Six dogs (group 1) underwent 1-week LL-VNS of the left cervical vagus nerve. Five normal dogs (group 2) were used as controls. SK2 protein levels were examined by using Western blotting. The ratio between SK2 and glyceraldehydes-3-phosphate-dehydrogenase levels was used as an arbitrary unit (AU). Results: We found higher SK2 expression in group 1 (0.124 ± 0.049 AU) than in group 2 (0.085 ± 0.031 AU; P<.05). Immunostaining showed that the density of nerve structures stained with SK2 antibody was also higher in group 1 (11,546 ± 7,271 μm(2)/mm(2)) than in group 2 (5321 ± 3164 μm(2)/mm(2); P<.05). There were significantly more ganglion cells without immunoreactivity to tyrosine hydroxylase (TH) in group 1 (11.4%±2.3%) than in group 2 (4.9% ± 0.7%; P<.05). The TH-negative ganglion cells mostly stained positive for choline acetyltransferase (95.9% ± 2.8% in group 1 and 86.1% ± 4.4% in group 2; P = .10). Immunofluorescence confocal microscopy revealed a significant decrease in the SK2 staining in the cytosol but an increase in the SK2 staining on the membrane of the ganglion cells in group 1 compared to group 2. Conclusions: Left LL-VNS results in the upregulation of SK2 proteins, increased SK2 protein expression in the cell membrane, and increased TH-negative (mostly choline acetyltransferase-positive) ganglion cells in the left stellate ganglion. These changes may underlie the antiarrhythmic efficacy of LL-VNS in ambulatory dogs.