Electrophoresis Flashcards
What kind of technique is it?
Separation
What is it?
Movement of charged particles or ions within an electric field
Ions that differ in charge and/or mass will move at different rates.
If they have a common starting point some molecules will move faster than others.
How does it work?
If a voltage is applied between two electrodes an electric field (potential gradient) is produced.
E=voltage/ distance between electrodes =V/d (volts/cm)
Particle movement
Positive charged ions move to NEGATIVE (cathode) electrode (known as cations) (+)
Negative charged ions move to POSITIVE electrode (anode) known as anions
Movement of the ion is termed…
Electrical mobility
Used to seperate..
Amino acids, peptides, glycoproteins proteins, nucleotides and nucleic acids and sugars
Factors affecting mobility
Size and shape of the molecule and charge
Buffer pH
Medium
How is mobility affected by charge…
Net charge of ion will determine which direction it will move
And how much friction from the medium it encounters
How is mobility affected by molecule shape?
Structural differences
Globular will migrate differently to fibrous proteins
The supporting medium should be..
Inert but can show molecular sieving, absorption and electro osmosis
Examples of medium
Paper, acetate, agarose gel, polyacrylmide gel
What is molecular sieving?
Associated with gels that consist of pores
Pores restrict flow of ions esp large ones
Smaller ones do not experience resistence and travel further
Separation by molecular size
What is adsorbtion?
Rocket shape appearance on medium surface
Caused by retention of molecules and slows down migration of sample
What is electro osmosis?
Occurs in media that possesses negative charged groups on surface
Groups attract cations (-) present in buffer
Cations are hydrated
When electric field is applied cations and water are attracted to cathode opposite to flow to anode
Weakly charged ions carried to cathode too
Forms basis of capillarity electro phoresis
Reason for staining
Most proteins are colourless
