SDS‐Polyacrylamide gel electrophoresis (PAGE) and western blots were used to evaluate the phosphorylation of cAMP‐dependent protein kinase A (PKA) and phospholamban (PLN), two proteins related to intracellular calcium transients. The human heart lysates (20 μg) were separated via SDS‐PAGE, transferred to a PVDF membrane, and probed overnight with primary antibodies for GADPH (Merck, 8795,1:5000), Phospho‐Thr197/PKA (Cell‐Signaling Technology, 4781, 1:1000), PKA (Cell‐Signaling Technology, 4782,1:1000), and phospho‐Ser16/PLN (Badrilla, A010‐12AP, 1:3000). Bands were visualized using enhanced chemiluminescent ECL (Amersham ECLTM Primer Western Blotting Detection Reagent (GE Healthcare) (RPN2232) in a ChemiDoc XRS+system using Image Lab software from Bio‐Rad Laboratories.
Amersham ecltm primer western blotting detection reagent
Manufactured by GE Healthcare
Sourced in United States
The Amersham ECLTM Primer Western Blotting Detection Reagent is a chemiluminescent detection system for identifying and quantifying proteins in Western blot analysis. It provides a sensitive and reliable method for protein detection.
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2 protocols using amersham ecltm primer western blotting detection reagent
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Evaluating PKA and PLN Phosphorylation
2
Western Blot Protocol for Protein Analysis
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Left ventricular specimens from the border area or harvested un-treated and treated cells were collected, washed, and lysed with RIPA (THERMOFISHER, USA) supplemented with PMSF (MERCK, USA). The total protein was extracted and the protein concentration of the lysates was quantified by the BCA protein45 (link). Protein (35 μg) was denatured, separated by SDS-PAGE electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane (MERCK MILLIPORE, USA). The transferred membranes were blocked using 5% bovine serum albumin (BSA) in TBST and incubated with the primary antibodies summarized in additional file (Table S3).
Bands were visualized using enhanced chemiluminescent ECL (AMERSHAM ECLTM Primer Western Blotting Detection Reagent (GE HEALTHCARE, NJ, USA) (RPN2232)), using a ChemiDoc XRS + system with Image Lab software from BIO-RAD LABORATORIES (Berkeley, CA, USA).
Bands were visualized using enhanced chemiluminescent ECL (AMERSHAM ECLTM Primer Western Blotting Detection Reagent (GE HEALTHCARE, NJ, USA) (RPN2232)), using a ChemiDoc XRS + system with Image Lab software from BIO-RAD LABORATORIES (Berkeley, CA, USA).
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