Eletrophoretic techniques

What is electricity?

Flow of electric charge

What is an electric current?

Flow of electrons (negative) or rarely (positive) ions

What is potential difference (PD)

Work done per unit of charge between two points in an electric field.

Units of PD

Volts (V)

When does current flow?

When there is a PD between two points in electric field

What happens when power supply is disconnected?

No PD between electrodes

What happens when power supply is connected?

PD created between electrodes

What happens when PD is present?

Charged particles will flow between electrodes

Define electrophoresis

The movement or migration of a charged particle in an electric field

What type of process is electrophoresis?

Incomplete type of electrolysis (stops before molecules reach electrodes)

Which biological molecules are charged?

• Amino acids, peptides & proteins

• Nucleotides & nucleic acids

What determines charge?

Nature of electric charge is pH-dependent

What are cations?

Positively-charged molecules migrate to negative cathode

What are anions?

Negatively-charged molecules migrate to positive anode

Describe electrophoresis equipment

• Power pack (provides electrical power)

• Electrodes (anode and cathode)

• Electrophoresis unit (vertical or horizontal)

• Gel (support medium)

• Buffer solution (maintains pH and charge)

• Sample wells (hold samples)

What opposes movement?

Frictional resistance (frictional coefficient)

What affects friction?

size and shape

gel pore size

buffer viscosity

Define velocity of a molecule (formula)

velocity=potential difference X charge/distance

Why do molecules move at different speeds?

Different size

different speed—.different friction

What results from this?

seperation of molecules at different distances

DNA movement pattern

• Small fragments move fastest

• Large fragments move slowest

Why is heat generated?

gel resistance during electrophoresis

Problems caused by heat

• Increased diffusion → band broadening

• Convection currents → sample mixing

• Protein denaturation

• Reduced viscosity → variable velocity

• Temperature gradients → uneven bands

How is heat minimised?

Control voltage

What is agarose?

Linear polysaccharide

What does pore size depend on

concentration

Use of pore size

DNA and RNA electrophoresis

What is polyacrylamide?

Cross-linked polymer

Use

Protein separation

DNA buffer

TAE (TRIS-acetate + EDTA)

RNA buffer

TBE (TRIS-borate + EDTA)

SDS function

Denatures proteins and gives uniform negative charge

Loading dye contains

tracking dye

density agent

What is a ladder?

molecular weight marker

SDS-PAGE

Separates proteins by size

Native PAGE

Separates proteins by size and charge

Isoelectric focusing

Separates proteins by pI

2D electrophoresis

Separates by pI then size

DNA electrophoresis

Agarose gels, size-dependent separation

RNA electrophoresis

Used to assess RNA integrity