The cultured cardiomyocytes and heart tissues were washed twice with cold PBS, harvested, lysed in ice‐cold lysis buffer (pH = 7.4, 100 mM KCl, 10 mM HEPES, 1.5 mM MgCl), sonicated, homogenized and then subjected to protein extraction with RIRP buffer with a phosphatase inhibitor cocktail and a protease inhibitor cocktail. After centrifugation, a Pierce BCA protein assay kit was used to determine the protein concentration. Equal concentrations of soluble lysate protein (50 μg) were added to a 12% SDS‐PAGE Bis‐Tris gel, and the protein was transferred to immunoblot polyvinylidene fluoride (PVDF) membranes (Bio‐Rad). Membranes were incubated and blocked overnight at 4℃ with antibodies against p‐AMPK/AMPK, p‐Akt/Akt and GAPDH. Then, the membranes were incubated with anti‐rabbit horseradish peroxidase–conjugated secondary antibodies (1:10 000 dilution). The immunoreactive bands were analysed with an enhanced chemiluminescence substrate kit (Biological Industries, BI).
Enhanced chemiluminescence substrate kit
Manufactured by Sartorius
Sourced in Israel
The Enhanced Chemiluminescence Substrate Kit is a laboratory reagent that enables the detection and quantification of proteins through the process of chemiluminescence. It provides a sensitive and reliable method for visualizing and analyzing protein samples.
Automatically generated - may contain errors
4 protocols using enhanced chemiluminescence substrate kit
1
Western Blot Analysis of Cardiac Proteins
2
Western Blot Analysis of Cardiomyocytes
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Cardiomyocytes were washed twice with cold PBS, harvested, lysed in ice-cold lysis buffer (pH=7.4, 100 mM KCl, 10 mM HEPES, 1.5 mM MgCl), sonicated and homogenized in RIRP buffer. After centrifugation, a Pierce BCA protein assay kit was used to determine the protein concentration. Equal concentrations of soluble lysate protein (50 mg) were added to a 12% SDS-PAGE Bis-Tris gel, and the protein was transferred to immunoblot polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA). Membranes were incubated and blocked overnight at 4°C with antibodies against p-AMPK/AMPK, p-Akt/Akt and GAPDH. Then, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution). The immunoreactive bands were analyzed with an enhanced chemiluminescence substrate kit (Biological Industries, BI, Israel).
3
Western Blot Analysis of Cardiomyocyte Signaling
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Cardiomyocytes were washed twice with cold PBS, harvested, lysed in ice-cold lysis buffer (pH = 7.4, 100 mM KCl, 10 mM HEPES, 1.5 mM MgCl), sonicated and homogenized in RIRP buffer. After centrifugation, a Pierce BCA protein assay kit was used to determine the protein concentration. Equal concentrations of soluble lysate protein (50 mg) were added to a 12% SDS-PAGE Bis-Tris gel, and the protein was transferred to immunoblot polyvinylidene uoride (PVDF) membranes (Bio-Rad, USA). Membranes were incubated and blocked overnight at 4°C with antibodies against p-AMPK/AMPK, p-Akt/Akt and GAPDH.
Then, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution). The immunoreactive bands were analyzed with an enhanced chemiluminescence substrate kit (Biological Industries, BI, Israel).
Then, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution). The immunoreactive bands were analyzed with an enhanced chemiluminescence substrate kit (Biological Industries, BI, Israel).
4
Cardiomyocyte Protein Expression Analysis
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Cardiomyocytes were washed twice with cold PBS, harvested, lysed in ice-cold lysis buffer (pH = 7.4, 100 mM KCl, 10 mM HEPES, 1.5 mM MgCl), sonicated and homogenized in RIRP buffer. After centrifugation, a Pierce BCA protein assay kit was used to determine the protein concentration. Equal concentrations of soluble lysate protein (50 mg) were added to a 12% SDS-PAGE Bis-Tris gel, and the protein was transferred to immunoblot polyvinylidene uoride (PVDF) membranes (Bio-Rad, USA). Membranes were incubated and blocked overnight at 4°C with antibodies against p-AMPK/AMPK, p-Akt/Akt and GAPDH. Then, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution). The immunoreactive bands were analyzed with an enhanced chemiluminescence substrate kit (Biological Industries, BI, Israel).
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