Cell fractionation allows for the individual study of organelles to better understand their biochemical functions and the molecular basis of diseases. The protocol involves homogenizing cells in buffer to break them open followed by differential centrifugation to separate organelles based on density. The purity of isolated fractions is assessed using marker enzymes that are signature to specific organelles, such as DNA polymerase for the nucleus or catalase for peroxisomes.

1. CELL
FRACTIONATION

2. Why cell fractionation?
• Individual study of each organelle
• Biochemical functions studied
• Molecular basis of disease

4. Protocol
• Cell is homogenized in aqueous
buffer- mechanically or by Tween-
20 (a lipid solvent)
• Organelles are purified
• Isotonic 0.25 M sucrose solution
at pH 7.4- the process is carried
out
• Homogenate is filtered
• Differential centrifugation

5. Differential Centrifugation

6. Organelles of similar
sedimentation
Coefficient separated by
Isopyknic (same density)
centrifugation technique

7. Marker Enzymes
• Purity of isolated subcellular fraction is
assessed
• Signature of the specific organelle

8. SUBCELLULAR ORGANELLE MARKER ENZYMES
PLASMA MEMBRANE 5’ Nucleotidase, Na+-K+ ATPase
NUCLEUS DNA Polymerase, RNA polymerase
ER Glucose-6-phosphatase
Cytochrome-b5 reductase
GOLGI BODIES Galactosyl transferase
Mannosidase
LYSOSOMES Acid phosphatase
Cathepsin
β- Glucuronidase
MITOCHONDRIA Succinate dehydrogenase
Cytochrome C oxidase
Oligomycin -sensitive ATPase
PEROXISOMES Catalase
CYTOSOL LDH, G-6-PD
MARKER ENZYMES FOR SUBCELLULAR FRACTIONS

9. One thing you cannot recycle is TIME