Extracellular vesicles or whole cell samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immobilon FL (EMD Millipore). Blots were probed with antibodies to vacuolar H+-ATPase,17 (link) anti-Actin (Sigma, cat# ZMS1004), anti-CD81 (eBioscience clone, Eat-2) and anti-GP96 (Life Technologies, 36-2600). These were then probed with horse-radish peroxidase secondary antibodies (Sigma). We used a chemiluminescent substrate (ThermoFisher; Super Signal West Pico) and a BioRad ChemiDoc MP (BioRad) to detect bands. The Optimal Autoexposure setting was used to acquire images. The raw chemiluminescent data were minimally processed using brightness and contrast controllers equally over the entirety of each blot in Adobe Photoshop for final figures.
Horse radish peroxidase secondary antibodies
Manufactured by Merck Group
Horse-radish peroxidase (HRP) secondary antibodies are enzyme-labeled antibodies used for detection in various immunological assays. They are conjugated with the enzyme horseradish peroxidase, which catalyzes a colorimetric or chemiluminescent reaction for signal amplification. HRP secondary antibodies are commonly used in techniques such as ELISA, Western blotting, and immunohistochemistry.
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Lab products found in correlation
Anti actin, by Merck Group (1 mentions)
Anti cd81, by Thermo Fisher Scientific (1 mentions)
Immobilon fl, by Merck Group (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Supersignal west pico, by Bio-Rad (1 mentions)
Goat anti mouse iga, by Merck Group (1 mentions)
Goat anti mouse igm, by Merck Group (1 mentions)
2 protocols using horse radish peroxidase secondary antibodies
1
Extracellular Vesicle Protein Analysis
2
Quantification of Anti-E7 Immunoglobulin Titers
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The sera from each group of immunized mice were pooled and analyzed after the second and third dose of immunogens. To determine the anti-E7 specific immunoglobulin (Ig)G titer, the sera pools were serially diluted (two-fold and tenfold) and assayed by enzyme-linked immunosorbent assay.25 (link) The endpoint dilution corresponded to an optical density absorbance <0.1 at 450 nm. Sera pools diluted 1:100 were used to analyze anti-E7 IgM, IgA and IgG isotypes (IgG1, IgG2b, IgG2c, and IgG3). Antigen–antibody complexes were detected using the following horseradish peroxidase secondary antibodies (Sigma-Aldrich): rabbit anti-mouse IgG (H + L), goat anti-mouse IgM (μ-chain specific), goat anti-mouse IgA (α-chain specific), and goat anti-mouse IgG1, IgG2b, IgG2c, and IgG3. Horseradish peroxidase activity was revealed using tetramethyl benzidine in the presence of H2O2. After 30 minutes at room temperature, the enzymatic reaction was stopped by adding 1 M sulfuric acid (50 μL per well). Washing steps were done using 400 μL per well of phosphate-buffered saline containing 0.05% Tween-20 in an automatic washer.
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