Cardiac homogenates were prepared in 300 µl of 10 mM NaHCO3 and 100 µl 20% SDS (sodium dodecyl sulfate). Mixtures were kept at 25°C for 30 min before centrifugation to remove debris. Thereafter, supernatants (called homogenates) were kept at −20°C until further analysis. Western blot analysis was performed as reported (Gergs et al., 2004 (link)). Aliquots of 100 µg of protein were loaded per lane. The antibodies against ERK (extracellular regulated kinase), AKT (protein kinase B), phospho-ERK, phospho-AKT, and A2A-ARs were obtained from Merck (Darmstadt, Germany). All secondary antibodies were conjugated with an alkaline phosphatase (Sigma-Aldrich, Taufkirchen, Germany). Bands were detected using enhanced chemifluorescence (GE Healthcare, Freiburg, Germany), and fluorescent bands were visualized in a Typhoon 9410 PhosphorImager and quantified using the ImageQuaNT software (GE Healthcare, Freiburg, Germany). enhanced chemifluorescence detection was carried out according to the manufacturer’s instructions (GE Healthcare, Freiburg, Germany).
Enhanced chemifluorescence
Manufactured by GE Healthcare
Sourced in Germany
Enhanced chemifluorescence is a highly sensitive method for detecting and quantifying proteins and nucleic acids in various applications, such as Western blotting and gene expression analysis. It utilizes a chemiluminescent substrate that emits light upon reaction with the target analyte, which is then detected and measured by specialized imaging equipment.
Automatically generated - may contain errors
2 protocols using enhanced chemifluorescence
1
Western Blot Analysis of Cardiac Signaling
2
Protein extraction and analysis from human keratinocytes
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Human primary keratinocytes were washed twice with ice-cold phosphate-buffered saline, scraped from the dishes in ice-cold whole-cell extraction buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.2, 75 mM NaCl, 2.5 mM MgCl2, 0.5 mM dithiothreitol, 0.2 mM EDTA, 20 mM β-glycerophosphate, 0.1% Triton X-100) supplemented with 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Equal amounts of whole-cell lysate were subjected to SDS–PAGE and transferred to Immobilon-P filter paper (Millipore, Bedford, MA). Immunoreactive proteins were visualized by enhanced chemifluorescence according to the manufacturer's protocol (GE Healthcare, Piscataway, NJ).
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