Electrophoresis – Questions & Solutions (Graded A+)
Electrophoresis: def. Right Ans - migration of charged particles in an
electrical field
Zone Electrophoresis Right Ans - performed on pourous medium (agarose
gel), results in separation of particles based upon size and charge
-bands of particles can be visualized, and quantified
-used in the clinical lab to separate proteins and other molecules in clinical
samples
e.g. serum, urine, CSF, and other fluids
-separating serum proteins, lipoproteins, glycoproteins, and isoenzymes
Separation Right Ans - migration is the distance the molecules moves from
the point of application
Rate of migration is dependent on: Right Ans - 1) net electric charge
2) size and shape
3) electric field strength
4) support medium
5) temperature
Electrophoretic Mobility Right Ans - -the velocity that a particle moves
through a support medium in an electric field, dependent on size and charge
-directly proportional to net charge, and indirectly proportional to size
Basic Electrophilic System Right Ans - 1) Support Medium: usually a type of
gel, such as agarose, sample is applied to gel
2)Buffer: electrolyte solution that has two functions, a)fix the pH of the
process
b)carry electric current
3)Chamber with electrodes: filled with buffer and gel placed inside, two
electrodes, anode (+) and Cathode (-)
4)Power Supply: provide current to system to establish electrical field
-current and voltage can be regulated
, Basic Electrophoresis Process Right Ans - 1)Sample applied to gel at
specific location
2)gel placed in chamber filled with buffer
3)electric current applied by power field
4)proteins migrate in the electrical field, after specified time period, power is
turned off, and gel is removed
5)the gel is dried or fixed, then stained to visualize separated bands
Factors affecting Separation: pH (protein charge) Right Ans - -
ampholytes/zwitterions: proteins are amphoteric, are either (+) or (-)
charged
-net charge is dependent on the pH of the solution they are in
-therefore electromorphic mobility is impacted by the pH of the buffer
-charge is dependent on the relative number of amino and carboxyl groups
-in acid pH, amino groups are (+)
-in basic pH, amino groups are (-)
pH (isoelectric point) Right Ans - pI, the specific pH level that net charge on
protein is zero
-dependent on protein structure and amino acid composition
-the difference in the buffer pH, relative to the isoelectric point of the protein,
determines the magnitude and type of charge on the protein
- if pH of buffer = pI, the net charge is zero
-if pH less than pI, the net charge is positive, cation migrates to the cathode
-if pH greater than pI, the net charge is negative, anion migrates to the anode
pH general Right Ans - -mostly done with buffer at pH 8.6
-serum proteins will be negatively charged anions and migrate towards the
anode
-magnitude of difference btw pI and pH of buffer determines the magnitude of
charge on a protein,
the greater the net charge (the further pI and pH are apart), the greater the
mobility
Ionic Strength Right Ans - increased [electrolytes] in buffer, is an increase
in ionic strength
-as proteins move through the buffer, they collect clouds of electrolyte ions
from the buffer
Electrophoresis: def. Right Ans - migration of charged particles in an
electrical field
Zone Electrophoresis Right Ans - performed on pourous medium (agarose
gel), results in separation of particles based upon size and charge
-bands of particles can be visualized, and quantified
-used in the clinical lab to separate proteins and other molecules in clinical
samples
e.g. serum, urine, CSF, and other fluids
-separating serum proteins, lipoproteins, glycoproteins, and isoenzymes
Separation Right Ans - migration is the distance the molecules moves from
the point of application
Rate of migration is dependent on: Right Ans - 1) net electric charge
2) size and shape
3) electric field strength
4) support medium
5) temperature
Electrophoretic Mobility Right Ans - -the velocity that a particle moves
through a support medium in an electric field, dependent on size and charge
-directly proportional to net charge, and indirectly proportional to size
Basic Electrophilic System Right Ans - 1) Support Medium: usually a type of
gel, such as agarose, sample is applied to gel
2)Buffer: electrolyte solution that has two functions, a)fix the pH of the
process
b)carry electric current
3)Chamber with electrodes: filled with buffer and gel placed inside, two
electrodes, anode (+) and Cathode (-)
4)Power Supply: provide current to system to establish electrical field
-current and voltage can be regulated
, Basic Electrophoresis Process Right Ans - 1)Sample applied to gel at
specific location
2)gel placed in chamber filled with buffer
3)electric current applied by power field
4)proteins migrate in the electrical field, after specified time period, power is
turned off, and gel is removed
5)the gel is dried or fixed, then stained to visualize separated bands
Factors affecting Separation: pH (protein charge) Right Ans - -
ampholytes/zwitterions: proteins are amphoteric, are either (+) or (-)
charged
-net charge is dependent on the pH of the solution they are in
-therefore electromorphic mobility is impacted by the pH of the buffer
-charge is dependent on the relative number of amino and carboxyl groups
-in acid pH, amino groups are (+)
-in basic pH, amino groups are (-)
pH (isoelectric point) Right Ans - pI, the specific pH level that net charge on
protein is zero
-dependent on protein structure and amino acid composition
-the difference in the buffer pH, relative to the isoelectric point of the protein,
determines the magnitude and type of charge on the protein
- if pH of buffer = pI, the net charge is zero
-if pH less than pI, the net charge is positive, cation migrates to the cathode
-if pH greater than pI, the net charge is negative, anion migrates to the anode
pH general Right Ans - -mostly done with buffer at pH 8.6
-serum proteins will be negatively charged anions and migrate towards the
anode
-magnitude of difference btw pI and pH of buffer determines the magnitude of
charge on a protein,
the greater the net charge (the further pI and pH are apart), the greater the
mobility
Ionic Strength Right Ans - increased [electrolytes] in buffer, is an increase
in ionic strength
-as proteins move through the buffer, they collect clouds of electrolyte ions
from the buffer
