Module 11 Flashcards
(30 cards)
What is zone electrophoresis?
Separation of molecules based on size and charge on a porous support medium.
Bands of particles are visualized and quantified.
What is migration?
The distance molecules move from the point of application.
What is migration rate dependent on?
Net charge of molecule
Size and shape of molecule
Electric field strength
Support medium
Temperature
What is mobility?
The velocity that a particle moves with through a support medium in an electric field.
What is mobility proportional to?
Directly- net charge
Indirectly- size
What are the components of an electrophoretic system?
Support medium
Buffer
Chamber with electrodes (anode and cathode)
Power supply- provides current
What are the functions of buffer?
Fix pH of process
Carry electric current
What is the process of electrophoresis?
Sample is applied to gel
Gel is placed in chamber with buffer
Current is applied
Proteins migrate for a specified time
Gel is dried or fixed and then stained
What factors affect separation?
pH
Ionic strength
Voltage and current
Support media
How does pH affect separation?
Net protein charge is dependent of pH.
Acid- amino group is positively charged, migrated to cathode
Base- carboxyl group is negatively charged migrates to anode.
What is the isoelectric point and how does it affect separation?
pH at which the net charge of protein is zero.
Difference in buffer pH relative to pI of protein determines direction and magnitude.
pH>pI- positive charge, to cathode
pH
What is the normal movement of proteins?
Buffer pH~8.6
Protein pH~4.9-5.9
Therefore negatively charged, move to anode.
How does ionic strength affect separation?
Proteins collect clouds of electrolyte ions from the buffer.
Affects migration.
Higher- low mobility, small migration, high heat production (increased wick flow), high resolution.
Lower- high mobility, large migration, low heat production, low resolution
What buffers are typically used?
Barbital
Tris-boric acid EDTA
How does voltage and current affect separation?
Application of voltage produces an electric field.
Resistance is the opposition of current trying to flow.
Heat is produced by the movement of electrons against resistance.
Heat reduces resistance.
V = IR
How can voltage and current be altered to address heat production?
Constant voltage- as resistance decreases, current must increase to maintain voltage, faster migration rate
Constant current- resistance decreases, voltage must decrease to maintain current, no change in migration rate
Why is agarose gel a good support medium?
Low affinity for proteins.
Free of ionizable groups.
Naturally clear after drying.
What are limitations/sources of problems with electrophoresis?
Wick flow
Electroendosmosis
What is wick flow?
Heat produced causes solvent evaporation.
Draws buffer into support from both ends.
Buffer flow affects rate and length of migration.
How is wick flow avoided?
Ensure lid is closed
Cooling the support
What is electroendosmosis?
Chemical groups in the support absorb OH ions giving it a negative charge.
Positive ions cluster around the negative charges.
When current is applied the positive clouds move to the cathode causing solvent to flow with it.
Macromolecules may be forced to remain immobile or swept towards the cathode.
How can electroendosmosis be limited?
Using media with minimal surface changes or ionizable groups.
What is the purpose of staining?
Used to visualize and identify the different bands/zones.
What dyes are proteins stained with?
Coomassie blue
Amido black
