Cellular Fractionation

1. Subcellular fractionation
of organelles and markers

2. Animal Cell and organelles
Albert Claude in 1930 developed the technique of cell
fractionation & identified the different organelles. He
received Nobel Prize for the same in 1974.

3. Introduction
Cells contain organelles which perform a variety of specific functions.
 To obtain precise information about the structure and functions of
subcellular organelles, it is necessary to isolate them free from
contaminating organelles.
Hence subcellular fractionation using centrifugation technique is used.
Individual organelle is identified using specific markers.
This information is used to study potential cellular irregularities and
methods to correct them.

4. How is subcellular fractionation done?
This involves two steps
1. Tissue/cell homogenization
The tissue is crushed between two rotating plates or filtered through a
membrane or grinding with glass beads. Then the homogenate is run at low
speed to remove intact cells present in the supernatant.
Differential centrifugation
2. Separation of organelles
Density equilibrium centrifugation.

5. Subcellular fractionation – differential
centrifugation

6. Cellular Markers
• These are enzymes specific to certain organelles.
• Following the activity of these enzymes, it is possible to locate
the organelles.
• Presence of unrelated enzyme activity confirms presence of
other organelle contamination.
• However electron microscopy is the final step to determine
purity of the preparation and the presence of organelle in the
sample.

7. Common markers for organelles
Organelle Marker enzyme
Nucleus Lamin A & C
Mitochondria -Inner
memb
ATP synthetase
Mitochondria- outer
membrane
Monoamine oxidase
Mitochondria - matrix Citrate synthase
Lysosome Cathepsin
Golgi bodies Galactosyl transferase
Microsome Glucose -6-phosphatase
Cytoplasm Lactate dehydrogenase
Peroxisomes Catalase

8. Assay for cellular markers
C - Cytoplasmic
M- Membrane bound
N - Nuclear

9. References
• Sequential fractionation and isolation of subcellular proteins from
tissue and cultured cells.
www.sciencedirect.com/science/article/pii/S2215016115000564.
Science Direct by S Baghirova - 2015.Methods X 2(2015),440-445.
• Padh, H. 1992. Organelle isolation and marker enzyme assay. Pages
129–146, in Tested studies for laboratory teaching, Volume 13 (C. A.
Goldman, Editor). Proceedings of the 13th Workshop/Conference of
the Association for Biology Laboratory Education (ABLE), 191 pages.
• Text Book of Biochemistry. Vasudevan and Sreekumari.8th Edition
Page 10.

10. Thank youThank you