Luminata crescendo western horseradish peroxidase hpr substrates

Following treatment, cells were lysed on ice in a buffer consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton x-100, 1% sodium deoxcycholate, 0.1% SDS, phosphatase inhibitor (P5726, Sigma Aldrich), protease inhibitor (P8340, Sigma Aldrich), and phenylmethylsulfonyl fluoride (PMSF, P7626, Sigma Aldrich) for 30 minutes. Lysates were centrifuged at 14 000 rpm for 30 minutes at 4°C. Protein concentrations were determined using a Micro BCA^™ Protein Assay Kit (Thermo Fisher Scientific). Proteins were separated on SDS-PAGE gels by electrophoresis and transferred to Immobilon^®-P polyvinylidene fluoride (PVDF) transfer membrane (EMD Millipore). Precision Plus Protein Kaleidoscope Standards (161–0375, Bio-Rad, Hercules, CA) molecular weight markers were used to confirm expected size of target proteins. Antibodies were used per the manufacturers’ recommended protocol. Samples were visualized by enhanced chemiluminescence (ECL) using Luminata Classico or Luminata Crescendo Western horseradish peroxidase (HPR) substrates (EMD Millipore). Anti-β-actin or vinculin served as an internal control to ensure equal protein loading.

Briefly, cells were lysed on ice for 30 minutes in a buffer consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton x-100, 1% sodium deoxcycholate, 0.1% SDS, phosphatase inhibitor (P5726, Sigma Aldrich), protease inhibitor (P8340, Sigma Aldrich), and phenylmethylsulfonyl fluoride (PMSF, P7626, Sigma Aldrich). The lysates were then centrifuged at 14 000 rpm for 30 minutes at 4 °C. Protein concentrations were determined using a Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL), separated by electrophoresis on SDS-PAGE gels, and transferred to Immobilon®-P polyvinylidene fluoride (PVDF) transfer membrane (EMB Millipore). Precision Plus Protein Kaleidoscope Standards (161-0375, Bio-Rad, Hercules, CA) were used for molecular weight markers to confirm expected size of target proteins. Antibodies were used in accordance with the manufacturers’ recommended protocol. Samples were visualized by enhanced chemiluminescence (ECL) using Luminata Classico and Luminata Crescendo Western horseradish peroxidase (HPR) substrates (EMD Millipore). Anti-β-actin was used an internal control to ensure equal protein loading between samples.