Separation techniques

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Presentation transcript:

Separation techniques

Centrifugation DIFFERENTIAL CENTRIFUGATION DENSITY GRADIENT CENTRIFUGATION

DIFFERENTIAL CENTRIFUGATION

DENSITY GRADIENT CENTRIFUGATION

Meselson-Stahl Experiment

Gel Electrophoresis

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

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

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

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)

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

SDS Polyacrylamide Gel Electrophoresis (SDS PAGE)

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