Western blot assay was performed as described previously4 (link),36 (link). Briefly, cells at 70%–80% confluency were scraped with 1X RIPA cell lysis buffer (1 mM β-glycerophosphate, 20 mM Tris-HCl (pH 7.5), 1 mM Na2EDTA, 1 mM Na3VO4, 150 mM NaCl, 1 mM EGTA, 2.5 mM Na4P2O7, 1 μg/mL leupeptin, 1% Triton, 10% protease inhibitor). Proteins were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore). Immune-reactive bands were visualized using a Pierce enhanced chemiluminescence (ECL) detection kit (Thermo Scientific); β-actin was used as a loading control. Information pertaining to antibodies used is detailed in Supplementary Table 1 .
Pierce enhanced chemiluminescence ecl detection kit
Manufactured by Thermo Fisher Scientific
The Pierce enhanced chemiluminescence (ECL) detection kit is a lab equipment product designed for the detection of proteins in Western blotting applications. It utilizes a chemiluminescent substrate to enable sensitive and quantitative protein detection.
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Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Anti β actin monoclonal antibody, by Merck Group (1 mentions)
Metformin 1 1 dimethylbiguanide hydrochloride, by Merck Group (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Immobilon p polyvinylidene fluoride pvdf transfer membrane, by Merck Group (1 mentions)
Anti mouse igg fab specific peroxidase antibody, by Merck Group (1 mentions)
Anti rabbit igg whole molecule peroxidase antibody, by Merck Group (1 mentions)
3 protocols using pierce enhanced chemiluminescence ecl detection kit
1
Western Blot Assay Protocol
2
Western Blot Assay Protocol
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Western blot assay was performed as described previously (4, 36 (link)). Briefly, cells at 70% to 80% confluency were scraped with 1X RIPA cell lysis buffer [1 mmol/L β-glycerophosphate, 20 mmol/L Tris-HCl (pH 7.5), 1 mmol/L Na2EDTA, 1 mmol/L Na3VO4, 150 mmol/L NaCl, 1 mmol/L EGTA, 2.5 mmol/L Na4P2O7, 1 μg/mL leupeptin, 1% Triton, 10% protease inhibitor]. Proteins were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore). Immune-reactive bands were visualized using a Pierce enhanced chemiluminescence (ECL) detection kit (Thermo Fisher Scientific); β-actin was used as a loading control. Information pertaining to antibodies used is detailed in Supplementary Table S1.
3
Metformin-Based Protein Analysis Protocol
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