Western blot was performed for the analysis of expression of various apoptotic markers like p53wt, p53mut, Bcl-2 and Bax in tumor tissues of the treated groups42 (link). Before resolving expressed protein employing SDS-PAGE, protein content of each sample was estimated using a Bicinchoninic Acid solution kit (Sigma-Aldrich, Co., USA)43 (link). Briefly protein (30 µg/lane) was loaded and resolved by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto PVDF membrane. After blocking in 5% non-fat dry milk prepared in phosphate-buffered saline (PBS) with Tween-20 (PBS-T), the membrane was washed three times with PBST and incubated for 2 hrs at 37 °C with rabbit anti-mouse p53wt, rabbit anti-mouse p53mut, rabbit anti-mouse Bax and rabbit anti-mouse Bcl-2 primary antibodies. After incubation and stipulated washing steps, the membrane was further incubated with HRP conjugated goat-anti-mouse secondary antibody (1:5000) for 1 hr at 37 °C. Blots were developed with the ECL (Enhanced chemi-luminescence) detection system (Bio-Rad). The pixel density of the bands was quantified using GS-800 Calibrated Imaging Densitometer (Bio Rad, India).
Bicinchoninic acid solution kit
Manufactured by Merck Group
Sourced in United States
The Bicinchoninic Acid (BCA) solution kit is a colorimetric assay used for the quantitative determination of total protein concentration. The kit contains the necessary reagents, including the BCA working reagent, to perform the protein assay. The BCA method is based on the reduction of copper ions (Cu2+) to cuprous ions (Cu+) by proteins in an alkaline medium, and the subsequent colorimetric detection of the cuprous ions using a reagent containing bicinchoninic acid.
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2 protocols using bicinchoninic acid solution kit
1
Quantitative Analysis of Apoptotic Markers
2
Western Blot Analysis of Protein Expression
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Keratinocytes treated with the indicated compounds were washed twice in ice-cold phosphate-buffered saline (PBS). Cells were then lysed in lysis buffer and protein concentrations were determined using a bicinchoninic acid solution kit (Sigma-Aldrich, St. Louis, MO, USA). Proteins were separated on SDS-polyacrylamide/bis-acrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore Co., Bedford, MA, USA). The membranes were blocked for 1 h in skim milk (BD Bioscience, Franklin lakes, NJ, USA). After blocking the membranes, they were rinsed with TBS-Tween 20 (TBS-T, 0.1% Tween 20). Next, the membranes were incubated with primary antibodies for 1 h at room temperature or overnight at 4℃. The membranes were then washed with TBS-T and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies. After extensive washing, immunoreactive bands were detected using Enhanced Chemi-Luminescence reagents (ATTO Corporation, Tokyo, Japan).
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