Proteins from exosome-rich fractions, myocardial tissues, or cells were extracted with Radio-Immunoprecipitation Assay (RIPA) buffer, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotting onto nitrocellulose membranes (Millipore). After being blocked with 5% skim milk for 1 hour, the membranes were incubated with primary antibodies against CD9, CD63, HSP70, CD81, Bcl-2, Bax, Cyclin D1, Wnt3a, Phospho-GSK-3β (Ser9) (p-GSK-3β), GSK-3β, β-catenin, and β-actin (Cell Signaling Technology, Danvers, MA) overnight at 4°C. Then the membranes were incubated with horseradish peroxidase–conjugated goat antirabbit IgG or goat antimouse IgG (Cell Signaling Technology) for 1 hour at room temperature. Proteins were visualized by the enhanced chemiluminescence detection system (Amersham, San Francisco, CA), and band intensities were quantified using ImageJ software from 3 independent results normalized by β-actin.
Horseradish peroxidase conjugated goat antirabbit igg or goat antimouse igg
Manufactured by Cell Signaling Technology
Sourced in United States
Horseradish peroxidase–conjugated goat antirabbit IgG or goat antimouse IgG is a secondary antibody reagent used to detect and quantify rabbit or mouse primary antibodies in immunoassays. The horseradish peroxidase enzyme conjugated to the secondary antibody acts as a reporter molecule that can be visualized using a suitable substrate.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence detection system, by Cytiva (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
β actin, by Cytiva (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Ndufa9, by Abcam (1 mentions)
P tsc2, by Cell Signaling Technology (1 mentions)
Eomes, by Cell Signaling Technology (1 mentions)
2 protocols using horseradish peroxidase conjugated goat antirabbit igg or goat antimouse igg
1
Exosome-derived Protein Analysis via Western Blot
2
Western Blot Analysis of Cellular Signaling
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T cells were lysed in Ripa buffer containing a protease inhibitor and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates were centrifuged at 4 °C at 12,000× g for 10 min, separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked with 5% BSA in PBS containing 0.05% Tween-20 and incubated with various antibodies recognizing pAMPKα1 (T172), pAMPKα1 (S485), pS6 (S235/236), peIF4E (S209), pULK1 (S555), ATG7, AQP9, PGC1α, TFAM, OPA1, pDRP1 (S616), ID2, ID3, cMyb, cMyc, Blimp1, T-bet, ZEB2, FOXO1, TCF1, TRAF6, pTSC2 (S1387), Eomes, HIF-1α and β-actin (Cell Signaling Technology) and the Complex I subunit NDUFA9 (Abcam, Cambridge, MA, USA). The membranes were imaged using a BioRad Chemidoc MP (Bio-Rad, Hercules, CA, USA) after a second incubation step with a horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Cell Signaling) secondary antibody [18 (link)].
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