Western blotting was performed as previously described (Mata-Greenwood et al. 2010 (link)). Briefly, protein extracts (30 µg) were prepared in a cold lysis buffer, heat denatured in a Laemmli buffer, separated on SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked in 5% non-fat dried milk in 0.05% TBST for 1 h, and then probed in primary rabbit anti-GR (Santa Cruz Biotechnologies), monoclonal anti-BAG1 (Santa Cruz Biotechnologies), monoclonal anti-HSP90 (BD Biosciences, San Jose, CA, USA), and rabbit anti-FKBP51 (Stressmarq, Victoria, BC, Canada), diluted in blocking buffer (1 µg/ml) overnight at 4 °C. After three 10 min washes with TBST, the membranes were incubated with secondary antibodies that were diluted at 1:2000. Bound antibodies were visualized using the chemiluminscence substrate (ThermoFisher Scientific, Carlsbad, CA, USA). The membranes were then probed with monoclonal anti-β-Actin (Ambion, Austin, TX, USA). Data is presented as protein levels relative to β-Actin levels.
Chemiluminscence substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Chemiluminescence substrate is a reagent used in analytical techniques to detect and quantify specific biomolecules, such as proteins or nucleic acids, through the generation of light. The substrate undergoes a chemical reaction that emits light, which can be measured and analyzed to provide information about the target analyte.
Automatically generated - may contain errors
2 protocols using chemiluminscence substrate
1
Western Blotting Protocol for Protein Analysis
2
Western Blotting Protocol for Protein Analysis
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Western blotting was performed as previously described (Mata-Greenwood et al. 2010 (link)). Briefly, protein extracts (30 µg) were prepared in a cold lysis buffer, heat denatured in a Laemmli buffer, separated on SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked in 5% non-fat dried milk in 0.05% TBST for 1 h, and then probed in primary rabbit anti-GR (Santa Cruz Biotechnologies), monoclonal anti-BAG1 (Santa Cruz Biotechnologies), monoclonal anti-HSP90 (BD Biosciences, San Jose, CA, USA), and rabbit anti-FKBP51 (Stressmarq, Victoria, BC, Canada), diluted in blocking buffer (1 µg/ml) overnight at 4 °C. After three 10 min washes with TBST, the membranes were incubated with secondary antibodies that were diluted at 1:2000. Bound antibodies were visualized using the chemiluminscence substrate (ThermoFisher Scientific, Carlsbad, CA, USA). The membranes were then probed with monoclonal anti-β-Actin (Ambion, Austin, TX, USA). Data is presented as protein levels relative to β-Actin levels.
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