Module 11: Electrophoresis Flashcards
What is electrophoresis
the migration of charged particles in an electric field
Zone electrophoresis
typical for clinical labs
Results in a separation of particles based upon size and charge
migration
the distance the molecules move from the point of application
Things that affect rate of migration (5)
net electric charge of molecule size and shape electric field strength support medium temperature
Electrophoretic mobility
the velocity that a particles moves through a support medium in an electric field
dependent on net charge and size
Electrophoretic mobility is directly proportionate to
net charge
Electrophoretic mobility is indirectly proportionate to
size
Basic electrophoretic system consists of (4)
Support medium (gel such as agarose; sample applied to gel) Buffer (electrolyte solution that fix pH of process and carry electric current) Chamber with electrodes (filled with buff and gel placed inside; 2 electrodes - the anode (POS charged) and cathode (NEG charged) Power supply (provide current to system for electrical field; current and voltage can be regulated)
Basic electrophoresis process (5)
Sample applied to gel at “point of application”
Gel placed in chamber filled with buffer
Electric current applied by power supply
Proteins migrate in the electrical field; after specific amount of time, power is turned off and gel is removed
Gel is dried/fixed and then stained to visualize separated bands
4 factors affecting separation
pH
Ionic Strength
Voltage and current
Support Media
How pH affects separation
Proteins are amphoteric or ampholytes (can be pos/neg charged)
Net charge of protein depends on pH of solution they are in
In acid pH, amino group becomes pos charged
In basic pH, amino group becomes neg charged
pI is dependent upon proteins structure and amino acid composition
When protein is placed in pH specific buffer, difference between buffer pH and pI of protein determines magnitude and type of charge on protein
In acid pH, proteins amino group becomes
positively charged
In basic pH, proteins carboxyl group becomes
negatively charged
Isoelectric point (pI)
the specific pH level that net charge on the protein is zero
When a protein is placed in a buffer with a specified pH
the difference in the buffer pH relative to the isolelectric point of the protein determines the magnitude and type of charge on the protein
If the pH of the buffer is the same as the pI, the net charge is
zero
If the pH of the buffer is LESS than the pI, net charge is
positive
The protein is a cation and migrates towards the negatively charge CATHODE
If the pH of the buffer is MORE than the pI, the net charge is
negative
The protein is an anion and migrates to the positively charged ANODE
***IMPORTANT
For most EP methods, the buffer has an alkaline/basic pH, usually 8.6, so serum proteins will be
negatively charged anions and migrate towards the anode
The further apart the buffer pH and pI,
the greater the net charge on the protein and therefore the greater its mobility
the higher the concentration of electrolytes in a buffer,
the higher the Ionic strength
As proteins move through the buffer, they collect “clouds” of
electrolyte ions from the buffer
High ionic strength
Collects larger clouds of electrolyte ions from buffer
Low motility
Small total migration
Higher heat production (may denature proteins and increase wick flow)
**High resolution (each band is more distinct)
Low ionic strength
Higher (faster) mobility
Larger total migration
lower heat production
lower resolution
Voltage and current
Ohm’s Law
E=IxR
Heat is produced due to movement of electrons against resistance BUT heat reduces the resistance (increases the conductance) by increasing rate of evaporation of water from support medium and increasing migration rate
2 ways voltage and current can be applied to address heat production
1) Constant Voltage
- as resistance decreases, current must increase to maintain constant voltage
- change in current flow = faster migration rate (affects separation pattern)
2) Constant current!
- as resistance decreases, voltage must decrease to maintain constant current
- no change in current = no change to separation pattern
With constant current, migration rate remains relatively
constant
Support Media
Usually agarose gel
- low affinity for proteins
- free of ionizable groups (less interference during procedure)
- Naturally clear after drying which provides for good quality densitometric scans
2 limitations in electrophoresis
wick flow
electroendosmosis
Wick flow
heat = evaporation of water from support medium
this draws buffer into the support from both ends (anode and cathode)
Flow of buffer from both directions affects rate and length of migration
How can wick flow be avoided
ensuring lid of EP chamber is in place and support is cooled during high-voltage applications
Electroendosmosis
In certain support media with chemical groups that absorb OH- ions that give the support media a net neg charge
Neg charges fixed on support medium attract positive ions from buffer
When current is applied, charges attached to support remain fixed but pos charged clouds of ions are free to move to cathode cause solvent to flow with it
Macro molecules in solution can only move towards anode IF they sufficiently charged or large enough. Otherwise they may remain immobile or be swept backwards towards the cathode
How can electroendosmosis be limited
by use of support media with minimal surface charges or ionizable groups
Staining
used to visualize and identify the different bands
Coomassie blue or Amido black usually used
SPE gel regions
Albumin a1 a2 Beta **Point of application** Gamma region (swept backwards by electroendosmosis)
Quantitation (densitometry)
electrophoretic support medium is passed through optical beam of densitometer
The absorbance of each fraction of a sample is measured and displayed on chart as peaks
Some EP systems use digital scanners
For a given peak, the area UNDER the peak is proportional to concentration of the sample fraction in that band
Albumin
fasting moving protein and therefore moves farthest towards the anode during EP
Globulins
alpha 1
alpha 2
beta
gamma
Cathodic migration
gamma globulins separates towards the cathode side of the point of application due to electroendosmosis
EP gels are examined for
increases and decreases in relative quantities of each band and the presence or absence of bands
liver disease and renal disorders show a decrease in
albumin
infections show broad increases in
the gamma region
M-peaks
separate distinct peaks in the gamma region
may represent monoclonal antibodies
may indicate multiple myeloma
If an M-peak shows up, follow up with an
IFE test
Error: incorrect buffer used
Appearance: unpredictable pattern
depends on pH of the buffer
Error: Gel not completely dry prior to staining
wherever gel was wet, it is permanently stained, therefore unable to determine pattern
