Subcellular Fractionation to Demonstrate Activation of Intrinsic Apoptotic Pathway.
Hussein Chehade1, Alexandra Fox1, Gil G Mor1
1Department of Obstetrics and Gynecology, C.S. Mott Center for Human Growth and Development, Wayne State University, Detroit, MI, USA.
Methods in Molecular Biology (Clifton, N.J.)
|May 25, 2021
Summary
Cellular protein translocation, like cytochrome C movement during apoptosis, impacts protein function. This study details methods for isolating cellular fractions to study protein movement and function.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Proteins are compartmentalized within cells based on function.
- Stimuli can trigger protein movement between cellular compartments, altering function.
- Mitochondrial cytochrome C translocation initiates apoptosis.
Purpose of the Study:
- To describe experimental methods for studying protein translocation.
- To provide a protocol for subcellular fractionation.
- To demonstrate purity and integrity assessment of isolated cellular fractions.
Main Methods:
- Subcellular fractionation of cell pellets.
- Isolation of mitochondrial and cytoplasmic fractions.
- Western blot analysis for fraction purity and integrity.
Main Results:
- Detailed step-by-step protocol for fractionation.
- Methods for assessing purity of mitochondrial and cytoplasmic fractions.
- Validation of protein integrity in isolated fractions.
Conclusions:
- Subcellular fractionation is a key technique for studying protein translocation.
- Accurate fractionation and purity assessment are crucial for understanding protein function.
- This protocol facilitates research into protein movement and its functional consequences.
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