Electrophoresis Flashcards
What is electrophoresis?
A separation technique based on the movement of charged molecules in an electric field.
Different molecules move at different rates through the gel based on what?
Their net charge, size, shape and electric field strength.
Name 3 applications of electrophoresis?
Separation of nucleic acids in research and genetic diagnostics
Separation of proteins in research and diagnostics
Separation of small charged molecules (amino acids, pharmaceuticals)
What was the very first electophoresis technique in the 1930s?
Separating plasma proteins by moving boundary electrophoresis carried out in a U-shaped tube. But it had low resolution due to diffusion and convection currents.
Explain the net charge in an elecrtophoresis set up and how this lets charged molecules move across the gel.
The anode (+ electrode) lets negatively charged molecules (anions) move towards it. The cathode (- electrode) lets positively charged molecules (cations) move towards it. Higher charged molecules move faster than smaller charged.
How does the size and shape affect the movement of molecules through the gel?
Smaller molecules (in size and shape) move faster through the gel than bigger ones.
Linear DNA/circular DNA move at different speeds.
Globular/fibrous proteins move at different speeds.
How does field strength (voltage) affect mobility of molecules?
Mobility increases with increasing field strength (until heating effects occur that reduce mobility).
What does the equation for electrophoretic mobility mean? (EM= Eq/r)
E= electric field strength
q= net charge of molecules
r= radius of molecules (size+shape)
the mass:charge ratio affects the movement of molecules based on field strength.
What reaction occurs during electrophoresis and how is it stopped?
Electrolysis occurs and is stopped by switching off the electric current before molecules reach the electrodes.
Heating effects occur during electrophoresis. Why is this a problem?
Problems:
Causes convection currents which can lead to zone broadening by increased diffusion of sample and buffer.
Heat denatures samples like proteins and enzymes
Heat reduces buffer viscosity leading to reduced frictional resistance.
How are heating effects in electrophoresis avoided?
Use a power pack that provides constant power
Use a cooling device like a water cooling system
Why are supporting media used and give examples of supporting media?
To minimize diffusion and convection problems.
Paper, starch, agarose, cellulose acetate and polyacrylamide.
Cellulose Acetate is a media type used. Describe some of its characteristics.
Less hydrophilic than cellulose so holds less water.
Allows reduced diffusion with increased resolution.
Uniform with large pores
No molecular sieving
Agarose Gel is a media type used. Describe some of its characteristics.
Made from seaweed.
A linear polysaccaride.
Low concentration gives large pores
High concentration gives smaller pores
Polyacrylamide Gel is a media type used. Describe some of its characteristics.
Made by cross-linking polymerised chains of acrylamide.
Pore size is determined by concentration of acrylamide.
Which supporting media type is used for ROUTINELY separating DNA fragments?
Agarose Gel Electrophoresis.
