Western blotting analysis was performed as described31 (link). MCF-7 cells were treated with Chaetocochins G in 0, 5, 10 and 20 μg/mL. After 48 h, cells were harvested and washed with ice-cold PBS and then lysed in ice cold RIPA lysis buffer for 30 min. The lysate was centrifuged at 12000 rpm for 15 min at 4°C and the supernatant was collected, the total protein concentration was determined using the BCA assay; the proteins were then dissolved in SDS sample buffer and denatured. Proteins were separated using 10% SDS–PAGE, and then transferred to PVDF (polyvinylidene fluoride) (Bio-Rad, Hercules, CA) membranes. After incubated overnight at 4°C with the respective primary antibodies, the horseradish peroxidase-conjugated secondary antibodies were added. Finally, the reactive band was identified using an enhanced chemiluminescent substrate to horseradish peroxidase.
Pvdf polyvinylidene fluoride
Manufactured by Bio-Rad
PVDF (polyvinylidene fluoride) is a type of fluoropolymer material. It is a durable, chemically resistant, and thermally stable polymer used in various applications, including laboratory equipment and scientific research.
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4 protocols using pvdf polyvinylidene fluoride
1
Western Blot Analysis of Chaetocochins G in MCF-7 Cells
2
Lonchocarpin Modulates Lung Cancer Cells
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Western blotting was performed according to the method described in previous research38 (link). Human lung cancer cells H292 were pretreated with DMSO (as a control) and lonchocarpin at 10 μM for 12 h, 24 h and 48 h. Then, the cells were harvested and then washed with PBS and lysed in a radio immune-precipitation assay buffer for 30 min. BCA protein assay kit (Byontime, Beijing, China) was used for determination of the total protein contents. And the protein samples were dissolved and seperated on 10% SDS-PAGE gels. After that the proteins were transferred to PVDF (polyvinylidene fluoride) (Bio-Rad, Hercules, CA) membranes and incubated sequentially with blocking buffer, primary antibodies, and horseradish peroxidase-conjugated secondary antibodies.
3
Western Blot Protein Analysis
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Standard western blot analysis was performed [23 (link)]. Briefly, Cell lysates were washed with phosphate buffered saline (PBS) and lysed in RIPA (radioimmunoprecipitation assay) buffer. Then lysates were centrifuged at 12000 g for 30 min at 4 °C. The Bio-Rad Protein Assay kit (Bio-Rad Laboratories) was used to determine the samples protein concentration according to the manufacturers’ recommendations. The lysates were dissolved in 5 × SDS sample buffer and denatured, then subjected to 6 % to 12 % SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) according to molecular weight and transferred onto PVDF (polyvinylidene fluoride) (Bio-Rad, Hercules, CA) membranes. Membranes were blocked for 1 h in 5 % dried milk in TBS/T at room temperature and incubated overnight at 4 °C with the primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized with enhanced chemiluminescent substrate (Amersham Biosciences Corp., Piscataway, NJ).
4
Analysis of Cell Apoptosis and Proliferation
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Western blotting was performed to analyse the expression level of proteins involved in cell apoptosis and proliferation. U87 cells were seeded at a density of 5×104 cells per plate and allowed to attach overnight. Cells were then treated with free luteolin, Luteolin/MPEG-PCL micelles at a concentration of 40 μg/ml, 20 μg/ml, 10 μg/ml, 5 μg/ml and EM for 48h. Cells treated with normal saline or blank micelles served as control groups. In the next step cells were lysed in RIPA (radioimmunoprecipitation assay) buffer. The protein extracts were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide) and transferred to PVDF (polyvinylidene fluoride) (Bio-Rad, Hercules, CA) membranes. The membranes were then probed with primary antibody against pro-caspase-9, cleaved-caspase-9, Bax, Bcl-2, MAPK, p-MAPK and β-actin followed by incubation with secondary antibodies. The blots were visualized by an enhanced chemiluminescence detection system (Amersham Biosciences Corp., Piscataway, NJ). This process was repeated in triplicate.
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