How to Use Western Blot for Apoptosis and Cell Death Studies
Jack (Jie) Huang MD, PhD
Chief Scientist I Founder/CEO I Visiting Professor I Medical Science Writer I Inventor I STEM Educator
Western blotting is an effective method for detecting markers of apoptosis and cell death, helping researchers quantify and evaluate the pathways by which cells die in response to treatment or disease. The following is a step-by-step guide to using western blotting for apoptosis and cell death studies.
1. Sample preparation
Samples are collected from cells treated with an apoptosis inducer and untreated control cells. Preparation of lysates with protease and phosphatase inhibitors is critical to preserve proteins and their post-translational modifications. Quantifying protein concentrations to ensure equal loading is critical for comparing levels of apoptosis markers between treated and control samples.
2. Key apoptosis markers
(1) Caspases: Caspase-3, caspase-8, and caspase-9 are central players in apoptosis. Cleaved (activated) caspase-3 is a hallmark of apoptosis, and western blotting can detect this specific form.
(2) PARP (poly ADP ribose polymerase): Cleavage of PARP by caspases indicates cell death, making it a reliable marker of apoptosis. Western blotting can show the appearance of cleaved PARP.
(3) Bcl-2 family proteins: Proteins such as Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) regulate apoptosis. Their expression levels or ratios can provide insight into the tendency of cells to survive or die.
3. Gel electrophoresis and protein transfer
Equal amounts of protein (20-50 μg) are added to each lane and separated by SDS-PAGE. The proteins are transferred to PVDF or nitrocellulose membranes for antibody detection.
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4. Antibody incubation and detection
The membrane is incubated with primary antibodies against apoptotic markers, such as cleaved caspase-3, cleaved PARP, Bax, or Bcl-2. After washing, enzyme-conjugated secondary antibodies are applied, and bands are visualized using chemiluminescent substrates.
5. Analysis
The intensity of the bands corresponding to apoptotic markers is quantified and normalized to a loading control (e.g., β-actin). Changes in the levels of cleaved caspase or PARP cleavage indicate apoptosis.
By tracking these markers using Western blotting, researchers can accurately assess apoptosis and cell death in response to various experimental conditions.
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References
[1] Gaspar Banfalvi, Apoptosis 2016 (https://doi.org/10.1007/s10495-016-1333-3)
[2] Goutam Dey et al., Scientific Reports 2017 (DOI:10.1038/s41598-017-17652-z)