Electrophoresis Flashcards
What is Electrophoresis?
The motion of charged particles in a colloid under the influence of an applied electric field
What are factors that influence migration?
- Size (radius)
- Shape
- Charge
- Viscosity of the colloid
- Electrical
- Field Strength (potential diff./ voltage)
- Temperature
- Gel Effects (e.g. endosmotic flow)
What are components of an Electrophoresis system?
- Power Supply
- Temperature Control
- Buffer
- Detection system
- Gel/Matrix
- Sample Applicator
How does the power supply influence the electrophoresis system?
- Higher voltages lead to faster migration and better turn around times
- Capillary electrophoresis uses huge voltages typically kV (i.e. 10-100 x mains)
- May require non-standard power supply
- High voltages can lead to current resistance
- Resistance generates HEAT which can denature molecules and influence migration
What are cooling systems used in Electrophoresis systems?
Needs strict temperature control
- Traditional gels = stirring block + cold room
- Capillary electrophoresis uses a ‘Peltier device’ (heat absorbed at junctions between materials)
What is the purpose of buffer in electrophoresis?
- Carries current, controls pH and molecular charge
- Can add other molecules to influence migration e.g. SDS (denatures), ampholytes (pH gradient)
- ‘Stacking’ at buffer boundary improves resolution.
What are types of gel or support matrices?
Several types available depending on type and size of molecule to be separated, resolution required Starch / Cellulose - brittle, need pre-soak • Agarose typically used for DNA • Polyacrylamide popular for proteins
How can gel or support matrices be influenced?
- Can add molecules (e.g enzyme substate) to allow detection or influence migration
What are features of Agarose gels?
- Complex polysaccharide derived from seaweed.
- Higher % agarose results in smaller pores (0.5-2%)
What are features of Polyacrylamide?
Chemically crosslinked chains of acrylamide and bisacrylamide. • Total % and ratio of acryl : bisacryl determines pore size
What are benefits and drawbacks of Agarose gels?
Benefits
- Cheap
- Non-Toxic
- Can prepare in-house
- Sets rapidly
- Ideal for longer DNA 50-20000bp
Drawbacks
- Non-uniform pore size
- Gels weak at low % agarose
- Gel brittle at higher % agarose
- No CE-marking
- May not set evenly
What are benefits and drawbacks of Polyacrylamide gels?
Benefits
- Uniform pore size, Reproducible results
- High resolving power
- Stronger, thinner gels dissipate heat better
- Chemically inert
- Ideal for short DNA (5-500 bp) and proteins
Drawbacks
- Acrylamide is neurotoxic
- Longer time to wait until set
- More expensive
- Need to buy gels in from a company
What sample types can be used for electrophoresis?
- Need charged molecules (alter pH or add SDS if not)
- DNA ideal due to native negative charge – may need to amplify, sonicate, digest, label etc.
- Proteins can be run natively (large complexes) or with SDS (adds -ve charge, ensures migration due to size only)
- For serum, avoid haemolysis, fibrinogen, contrast media and other interfering substances
- Urine / CSF samples may need to be pre-concentrated
How is Urine assessed?
- Ideally an early morning urine
- Assess concentration using urine creatinine
- Vivaspin® urine concentrators for dilute urine samples e.g. elderly patients on diuretics, Centrifuge sample
- Microfilter retains proteins and removes excess water
How is the sample applied to the Gel electrophoresis?
- Usually around 2-50μL sample
- Can be done manually with a loading dye
Most automated systems use either:
- (1) Electrokinetic Application where a small voltage applied to drive sample into buffer,
- (2) Hydrodynamic application where positive pressure applied ‘pushes’ sample into buffer
