Western blotting analysis was performed as per the manufacturer’s protocols. Total proteins from human samples were extracted by lysis buffer (Beyotime, Nanjing, China). Protein quality and quantity were determined with the Bradford dye-binding protein kit (Bio-Rad Laboratories, CA, USA). Afterward, a total of 40 μg protein was loaded onto a 10% SDS polyacrylamide gel and transferred to a PVDF membrane. The primary antibodies against P53 were purchased from Cell Signal Technology (Danvers, MA, USA). Primary antibody against GAPDH and secondary antibodies were obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). Immunoreactivity of proteins of each sample was determined by enhanced chemoluminescent autoradiography (Thermo Scientific) using a FluorChem Q system (Proteinsimple, Santa Clara, CA, USA). Blots were quantified using Quantity One (Bio-Rad Laboratories, CA, USA).
Enhanced chemoluminescent autoradiography
Manufactured by Thermo Fisher Scientific
Sourced in United States
Enhanced chemoluminescent autoradiography is a lab equipment product that utilizes chemiluminescence and autoradiography techniques to detect and analyze the presence and quantity of target molecules or proteins in a sample. It provides a sensitive and quantitative method for visualizing and measuring specific biomolecular interactions or the expression of proteins of interest.
Automatically generated - may contain errors
2 protocols using enhanced chemoluminescent autoradiography
1
Western Blot Analysis of p53 Expression
2
Western Blot Protein Analysis
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Total proteins were extracted using a RIPA lysis buffer (pH = 7.5, Beyotime Biotechnology, Nantong, China) to generate the whole protein lysate. An equal amount of 40 μg protein was loaded to each lane in a 12% SDS-PAGE gel. Proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane after electrophoresis. After blockade of nonspecific antigens with 5% skim milk, membranes were incubated with primary antibodies overnight at 4°C. A secondary antibody which recognizes the primary antibody was then added, and the immunoreactivity was determined with enhanced chemoluminescent autoradiography (Thermo Scientific, PA, USA). GAPDH was synchronously developed for loading control.
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