Module 11: Electrophoresis Flashcards
Principle of electrophoresis
Separation of proteins based on charge and size in an electrical field
Electrophoresis clinical uses
Separate serum proteins to identify protein disorders (multiple myeloma: m-peak in gamma region; liver cirrhosis: characteristic beta-gamma bridging)
Electrophoretic mobility
Velocity the protein migrates
Factors that affect rate of migration
Net charge on the molecule Size and shape of the molecule Electric field strength Properties of the support medium Temperature of the operation
Amphoteric
Can have either a positive or negative charge
Proteins are amphoteric depending on the pH of the solution they are in
Isoelectric point (pI)
pH where the net charge of the protein is zero
If pI > pH it will have a (pos/neg) charge and migrate towards the (anode/cathode)
Positive
Cathode
If pI is less than pH it will have a (pos/neg) charge and migrate towards the (anode/cathode)
Negative
Anode
In pH of 8.6, proteins are (anions/cations) and migrate towards the (anode/cathode)
Anions
Anode
Buffers function to:
Maintain pH
Conduct electrical current
Higher ionic strength buffers = (slower and shorter/faster and longer) overall migration and produce (better/worse) resolution
Slower and shorter
Better
Benefits of agarose gel for electrophoresis
Few ionizable groups therefore less interference due to electroendosmosis
Low protein affinity
Rate of migration is determined by the:
Current
Ohm’s Law
E = I x R Voltage = current (amps) x resistance (ohms)
Effects of constant voltage
As heat is generated, resistance decreases, results in increase in current therefore migration increases
Effects of constant current
As heat is generated, resistance decreases, voltage decreases, current remains constant, therefore migration is constant
Densitometer
Uses photometry to measure absorbance of bands after gel is dried and stained
Wick flow
Evaporation of buffer in the middle draws up more buffer from the ends of the gel
Controlled by keeping lid on system and cooling temperature
Electroendosmosis
Flow of buffer ions and solvent in opposite direction of protein migration
Buffer cations and solvent flow toward cathode, gamma globulins are moved towards cathode (cathodic migration)
How is electroendosmosis minimized?
Using media with few ionizable groups and low protein affinity
Electrophoretic mobility is directly proportional to the _____ and inversely proportional to the ______ of the molecule.
Net charge
Size
What is the advantage of using constant current over constant voltage?
Migration rate remains relatively constant
Increasing ionic concentration of the buffer (increases/decreases) migration?
Decreases
Increasing ionic concentration of the buffer (increases/decreases) resolution?
Increases
Increasing ionic concentration of the buffer (increases/decreases) heat generation?
Increases
Most plasma protein pIs:
4.9-5.9
