Equal amounts of protein (100 μg/lane) from cell lysates or culture medium were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) or Tricine–SDS‐PAGE and transferred to polyvinylidene difluoride membranes (Hybond‐P; GE Healthcare, Pittsburgh). The blots were probed with the appropriate primary antibody, followed by HRP‐conjugated anti‐rabbit IgG (Cell Signaling Technology). Protein bands were visualized using an enhanced chemiluminescence (ECL) detection method (Bio‐Rad, CA), and band intensity was analyzed with a densitometer (LAS‐4000; GE Healthcare). Each experiment was repeated at least three times. GAPDH, measured quantitatively using a GAPDH antibody (ab181602), was used as control.
Enhanced chemiluminescence ecl detection method
Manufactured by Bio-Rad
Enhanced chemiluminescence (ECL) detection method is a technique used to detect and visualize specific proteins in a sample. It utilizes a luminescent chemical reaction to generate light, which can be captured and measured to quantify the presence and abundance of the target proteins.
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2 protocols using enhanced chemiluminescence ecl detection method
1
Western Blot Protein Analysis Protocol
2
Quantitative Western Blot Analysis
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Cell lysates were obtained from cells that were extensively washed with PBS and lysed directly in cell lysate solution. Protein concentration was determined using the BCA Protein Assay Kit. Equal amounts of protein (100 μg/lane) from cell lysates or culture medium were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or Tricine-SDS-PAGE and transferred to polyvinylidene difluoride membranes (Hybond-P; GE Healthcare). Blots were probed with an appropriate primary antibody, followed by HRP-conjugated anti-rabbit IgG (Cell Signaling Technology, CST). Protein bands were visualized using an enhanced chemiluminescence (ECL) detection method (Bio-Rad), and band intensity was analyzed with a densitometer (LAS- 4000; GE Healthcare). Immunoreactive protein content of each sample was calculated based on a standard curve constructed using BSA. Each set of experiments was repeated at least 3 times to confirm the results. The level of GAPDH protein, measured by quantitative Western blotting using GAPDH antibody (ab181602), was used as an extraction and loading control.
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