How to Extract Small Intestine Proteins and Which Part to Use for ZO-1 Expression Analysis
Protein Extraction
1. Sample Preparation
Excise a designated section of the small intestine from the experimental animals and immediately immerse it in ice-cold physiological saline to remove residual luminal contents.
2. Tissue Homogenization
(1) Homogenize the intestinal tissue using a mechanical homogenizer. During this process, the tissue is physically disrupted to release intracellular proteins into the extraction buffer.
(2) Use an appropriate buffer, such as phosphate-buffered saline (PBS) supplemented with protease inhibitors, or a non-ionic detergent-based buffer containing Triton X-100, to prevent proteolytic degradation.
3. Centrifugation
Clarify the homogenate by centrifugation at high speed (e.g., 12,000 × g) at 4°C for 10–15 minutes to remove unbroken cells and tissue debris.
4. Protein Concentration and Purification
Collect the supernatant containing soluble proteins. At this stage, additional purification and concentration can be performed using methods such as ammonium sulfate precipitation, dialysis, or chromatography techniques (e.g., affinity or size-exclusion columns).
Store the extracted proteins at -80°C to preserve their structural integrity and prevent degradation.
Small Intestine Sample Sectioning
The small intestine comprises the duodenum, jejunum, and ileum. ZO-1 protein is expressed across epithelial cells throughout the small intestine, though expression levels may vary by region. For targeted experimental analysis, the jejunum is recommended, as it serves as the primary site for nutrient and water absorption and exhibits relatively uniform epithelial cell structure and function.
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