Homogenates containing about 5 μg of apoA-I protein were heated for 5 min at 95 °C, and subjected to one-dimensional electrophoresis (SDS-PAGE) using NuPAGE 4–12% BisTris polyacrylamide gel on a Novex Mini-Cell (Invitrogen). After electrophoresis, the gels were submitted to silver staining, or the proteins were transferred from gel to a nitrocellulose membrane using the Trans-Blot SD semi-dry transfer cell (Bio-Rad). The nitrocellulose membranes were incubated with antibody specific to detect apoA-I (goat anti-human apoA-I, 11A-G2b, Academy BioMedical). Each membrane was incubated with compatible secondary antibodies conjugated with peroxidase, developed using Lumi-Light Western blotting Substrate Reagents (Roche Applied Science), and detection using x-ray equipment.
Lumi light western blotting substrate reagents
Manufactured by Roche
The Lumi-Light Western blotting Substrate Reagents are a chemiluminescent detection system used in Western blotting analysis. The reagents contain the necessary components to generate a light signal upon reaction with the target protein, allowing for the visualization and quantification of specific proteins in a sample.
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Lab products found in correlation
2 protocols using lumi light western blotting substrate reagents
1
Western Blot Analysis of ApoA-I
2
Western Blot Analysis of Planarian Proteins
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Proteins were obtained by homogenization of 4 planarians in lysis buffer as described above. Protein concentration was measured by Bradford method. Two independent samples were processed for each experimental condition. Twenty micrograms of proteins for each sample were separated by SDS-PAGE and electro-transferred onto a nitrocellulose membrane. Membrane was incubated with rabbit H3P antibody (UPSTATE) or mouse Dj-PiwiA antibody (Yoshida-Kashikawa et al., 2007) , diluted 1:500 in 1% skimmed milk. After incubation with a 1:16000 dilution of anti-rabbit or mouse peroxidase-conjugate secondary antibody (Biovision), detection was performed using Lumi-Light Western blotting Substrate reagents (Roche).
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