Samples to be visualized on PAGE were mixed with three volumes formamide supplemented with 20 mM EDTA and then heated for 5 min to 90°C. Samples were then loaded and run into 8% PAGE (19:1 Acrylamide:bis). Post staining of the gels was performed using 100 mL of 1× WB (140 mM KCl, 1 mM MgCl2, 10 mM NaH2PO4 pH 7.2, 0.05% Tween-20) supplemented with 20 nM TO1-Biotin, and Mango NASBA bands were imaged on a GE AI600RGB imager as described elsewhere (Yaseen et al. 2019 (link)). Alternatively, gels were stained with 1× SYBR Safe stain otherwise under the same conditions.
Ai600rgb imager
Manufactured by GE Healthcare
Sourced in United States
The AI600RGB imager is a lab equipment product from GE Healthcare. It is designed to capture high-quality images for various applications. The core function of the AI600RGB imager is to provide accurate and reliable image acquisition.
Automatically generated - may contain errors
4 protocols using ai600rgb imager
1
NASBA Band Visualization on PAGE
2
Western Blot Analysis of Protein Expression
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BMECs were washed thrice with PBS and lysed in cold radioimmunoprecipitation assay lysis buffer containing a protease inhibitor cocktail (P8340, Sigma-Aldrich, St. Louis, MO, USA) on ice for 30 min. Lysates were centrifuged at 12,000× g for 15 min. Protein concentration in the supernatant was measured by the Bradford assay method. Proteins were then boiled for 10 min and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For gel electrophoresis, 15 µg of total protein extracts was separated on 12.5% SDS-PAGE gel and then transferred to an Immuno-Blot polyvinylidene fluoride (Millipore, Temecula, CA, USA) membrane through wet transfer apparatus (BioRad, Hercules, CA, USA) for 1.5 h, at a 300 mA constant current. The membrane was incubated for 1 h with 5% non-fat dairy milk, washed thrice for 10 min, and the primary antibodies were probed overnight at 4 °C. The membranes were washed and probed with horseradish peroxidase (HRP) goat anti-rabbit IgG antibody and HRP goat anti-mouse IgG antibody for 1 h at 25 °C. Thereafter, chromogenic-enhanced chemiluminescence was performed. An AI600RGB imager (GE, Fairfield, CT, USA) and ImageLab 6.1 software (BioRad, Hercules, CA, USA) were used to reveal and analyze the chemiluminescence signal.
3
Immunoblot Analysis of Adult Brain Tissue
4
Investigating EphA2 and EphA3 Downregulation
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The downregulation of EphA2 and EphA3 was investigated as described previously.43 (link) Western blots for testing the presence of receptors were also performed as described previously.9 (link) Three independent experiments were performed for each QUAD version and the EphA2 and EphA3 receptors. Density of immunoreactive bands was measured on an AI600 RGB imager (GE). Data from 3 independent experiments were normalized to the corresponding β-actin and analyzed in Prism GraphPad.
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