1. Separation techniques

2. Centrifugation DIFFERENTIAL CENTRIFUGATION
DENSITY GRADIENT CENTRIFUGATION

3. DIFFERENTIAL CENTRIFUGATION

4. DENSITY GRADIENT CENTRIFUGATION

5. Meselson-Stahl Experiment

6. Gel Electrophoresis

7. Definition
Electro = Charge + Phorsesis= Carry
Electrophoresis = Separation of charged molecules by differences in their rate of migration in an electric field.

8. The Components of The System
Molecules to be separated
Proteins
Nucleic Acids
Support medium
Gel (Starch, Polyacrylamide or Agarose)
Buffer System
DC Power Source

9. Common Support Media for Biological Molecules
Proteins
Native Gel (Acrylamide or Starch)
Denaturing (SDS) Gel (Acrylamide)
Nucleic Acids – DNA & RNA
Agarose Gel
Acrylamide Gel

10. Factors that Influence Mobility
Properties of the Molecules to be separated
Molecular size (MW)
Molecular shape
Molecular charge
Properties of the System
Electric field strength (V/cm)
Porosity of the support medium (% S)
Conductivity of the buffer (R)

11. These Factors Interact
Mobility is proportional to charge/MW.
Mobility is proportional to field strength.

12. SDS Polyacrylamide Gel Electrophoresis (SDS PAGE)

13. Separation of DNA Molecules in Agarose Gels
The phosphate groups bear negative charge at neutral pH (2 phosphates/BP)
Therefore mobility will be based on number of base pairs/molecule