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Rapid procedure for obtaining tracheal apical membranes
B Q Shen1, C M Yang, J H Widdicombe
1Cystic Fibrosis Research Center, University of California, San Francisco 94143.
The American Journal of Physiology
|August 1, 1991
Summary
This study successfully isolated pure apical membranes from cow tracheal epithelium using a novel two-step purification process. The resulting membranes showed high enrichment of alkaline phosphatase, a key apical marker.
Area of Science:
- Cell Biology
- Biochemistry
- Epithelial Physiology
Background:
- Studying the function of specific cellular components requires pure membrane fractions.
- Cow tracheal epithelium is a relevant model for respiratory research.
Purpose of the Study:
- To develop and validate a method for isolating pure apical membranes from cow tracheal epithelium.
- To characterize the purity and integrity of the isolated apical membranes.
Main Methods:
- A two-step purification protocol involving Mg aggregation and differential centrifugation.
- Ouabain-affinity chromatography was employed for further purification.
- Enrichment of apical membrane markers (alkaline phosphatase) and exclusion of other cellular markers were assessed.
Main Results:
- The purification process significantly enriched alkaline phosphatase activity (21-fold) in the isolated apical membranes.
- Ouabain-affinity chromatography removed Na-K-adenosinetriphosphatase while preserving alkaline phosphatase levels.
- Markers for nuclei, mitochondria, and basolateral membranes were effectively excluded, indicating high purity.
Conclusions:
- A robust method for isolating highly purified apical membranes from cow tracheal epithelium was established.
- The isolated membranes are suitable for studying apical membrane transport and function.
- The technique is also effective with primary cultures of tracheal epithelium.