Electrophoresis
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What is electrophoresis?
It is a technique that involves migration of charged solutes in a liquid medium when an electrical field is applied
What type of technique is electrophoresis?
A separation technique
Towards what direction do negatively charged particles move?
Towards the anode (positively charged electrode)
Towards what direction do the positively charged particles move?
Towards the cathode (negatively charged electrode)
Depending on what side chains are present, what type of charge do proteins have?
They are amphoteric (can cat both as cation or anion)
What is electrophoretic mobility?
It is the rate of migration of particles in cm/s per unit of field strength as volts/cm
What is the isoelectric point of a protein?
The pH at which that protein has no net charge
What is the isoelectric point of albumin?
4.9
What range do the isoelectric points of globulins fall?
4.0-7.3
What is the isoelectric point of hemoglobin?
~ 7
What charge does a protein become when buffer pH is above the isoelectric point of that protein?
The protein becomes negative and moves towards the anode
What is the typical pH of the protein electrophoresis buffer?
8.6
How is the carboxyl group of proteins when they’re negatively charged ,owing towards the anode?
The carboxyl group is ionized
What is migration rate of particles?
How quickly particles move toward an electrode.
What factors affect the migration rate of particles?
Net charge of particles (most important factor, bigger the charge, the faster the particle) Size and shape of molecules (bigger the size, the slower the particle) Electrical field (more current, faster particle) Ionic strength of buffer (greater ionic strength, faster particle) pH of buffer (lower pH of buffer, slower particle) Viscosity (thicker medium, slower particle) Temperature (higher temp, faster particle)
What does the net charge of a particle depend on?
On the buffer and the resultant pH set by the buffer
What can too much ionic buffer strength cause?
Increased heat and smearing
Why is care taken with heat sensitive analytes?
High temperature can denature the protein and DNA/RNA Bands will be smeared or gel will melt
What is the most common type of electrophoresis?
Zone electrophoresis
How does zone electrophoresis work?
ZE is the separation of zones into bands. It separates zones of protein that are distinct from each other.
Name the support media used in zone electrophoresis
Agarose Polyacrylamide Cellulose acetate gel
What electrophoresis is mostly seen in the clinical lab and what type is it?
Serum protein electrophoresis Semi-quantitative
List the electrophoresis instruments
Support medium Buffer Sample Power supply Dye/stain Dryer/scanner
What electrophoresis instrument is crucial for good separation of bands and why?
Buffer. It protects the same place, applies charge to it and carries current