Tumor tissue samples were homogenized in lysis buffer containing 0.6 % SDS, 10 mM Tris-HCl, pH 7.5, supplemented with a mixture of protease inhibitors (Roche), and phosphatase inhibitors (Thermo Scientific). Equal protein aliquots were subjected to SDS-PAGE (10% acrylamide) under reducing conditions, and proteins were transferred to a polyvinylidene difluoride membrane (Millipore). Membranes were blocked with 3% BSA for 1 h at room temperature and probed with the appropriate antibody, followed by horseradish peroxidase–conjugated secondary antibody (KPL) and a chemiluminescent substrate (Biological Industries). Band intensity was quantified by densitometry analysis using Scion Image software.
Chemiluminescent substrate
Manufactured by Sartorius
Sourced in United States
Chemiluminescent substrate is a laboratory reagent used to detect and quantify the presence of specific proteins or enzymes in a sample. It generates a luminescent signal that can be measured by specialized equipment, allowing for sensitive and quantitative analysis.
Automatically generated - may contain errors
3 protocols using chemiluminescent substrate
1
Quantitative Western Blot Analysis
2
Western Blot Analysis of MCF-7 Cells
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MCF-7 whole cell lysates were homogenized in lysis buffer containing 0.6% SDS, 10 mM Tris-HCl, pH 7.5, supplemented with a mixture of protease inhibitors (Roche) and phosphatase inhibitors (Thermo Scientific). Equal protein aliquots were subjected to SDS-PAGE (8% acrylamide) under reducing conditions and proteins were transferred to a polyvinylidene difluoride membrane (Millipore). Membranes were blocked with 3% BSA for 1 hour at room temperature and probed with the appropriate antibody, followed by horseradish peroxidase-conjugated secondary antibody (KPL) and a chemiluminescent substrate (Biological Industries).
3
Western Blot Protein Analysis
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Cell and tumor tissue lysates were homogenized in lysis buffer containing 0.6% SDS, 10 mM Tris-HCl, pH 7.5, supplemented with a mixture of protease inhibitors (Roche) and phosphatase inhibitors (Thermo Scientific, Waltham, MA, USA). Equal protein aliquots were subjected to SDS-PAGE (10% acrylamide) under reducing conditions and proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany). Membranes were blocked with 3% BSA for 1 h at room temperature and probed with the appropriate antibody, followed by horseradish peroxidase-conjugated secondary antibody (KPL, Milford, MA, USA) and a chemi-luminescent substrate (Biological Industries, Beit-Haemek, Israel). Band intensity was quantified by densitometry analysis using ImageJ software.
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