360 - Electrophoresis Flashcards
The rate of
migration of an analyte during electrophoresis is dependent on the properties of …
the support media,
the electrical field strength,
the temperature
T or F. DNA migration through agarose is considered a function of size, as all molecules have the same mass to charge ratio
T!
proteins and nucleic acids migrate through agarose gels based on their …
mass to charge ratio
why is agarose preferable over starch and cellulose acetate gels?
they are easy to handle, has no charge, and minimally contributes to electroendosmosis.
it also has low affinity for proteins and is clear when dried = documentation of densitometry
Describe electroendosmosis:
- media is usually negatively charged and counterions are cations
- counterions are hydrated
- electric field applied = charged media components stationary but counterions move towards oppositely charged electrode, taking water with them
- result = net movement of solvent in single direction
- strength of movement can slow down or reverse direction of analyte migration
This determines the net charge of the analytes during electrophoresis
the pH ofthe buffer
This determines the net charge of the analytes during electrophoresis
the pH of the buffer
T or F. Both protein and DNA electrophoresis are carried out at an acidic pH
F! ALKALINE
____ in the buffer carry the current
Ions
Under fixed current conditions, the rate of migration of macromolecules in a system _______________ as the ionic strength (conductivity) of the buffer increases
decreases
how do we keep the system at a constant current?
the voltage must be decreased which means the applied electrical field is decreased = less electrical force on the macromolecules
Under these conditions,
resolution decreases, bands broaden, as the macromolecules diffuse in the gel
To maintain constant voltage conditions, the current must be _________ as the ionic strength (conductivity)
of the buffer __________
increased; increases
migration velocity of the macromolecules is unchanged BUT
too high of a current may cause overheating
what can excessive heat lead to?
protein denaturation
can produce convection currents in the buffer, which warps the electrophoresis patterns
best to use a constant current supply to minimize the effects of heat on the system
how does pH affect electrophoresis?
determines the charge of analyte and therefore it’s mobility
how does ionic strength affect electrophoresis?
- alters voltage (in a constant current system)
- increased ionic strength usually reduces the migration rate
- increased ionic strength usually increases heating
how does current affect electrophoresis?
too much current results in excessive heat production
how does voltage affect electrophoresis?
migration rate is proportional to voltage
how does temperature affect electrophoresis?
Temperature gradients cause curved bands
Excess heat can denature proteins
Lower temperatures decrease migration rates
how does time affect electrophoresis?
band resolution increases with time
how does the suppot media affect electrophoresis?
Electroendosmosis and pore size affect migration rates
how does the support media affect electrophoresis?
Electroendosmosis and pore size affect migration rates
The rate migration of an analyte in electrophoresis is based on the analyte’s…
charge, molecular weight, and shape
T or F. In agarose gel electrophoresis, the pore size is not sufficiently small enough to allow for molecular sieving; thus, proteins and nucleic acid migrate based on their mass to
charge ratio
T!
DNA has a __________ charge
negative
- migrates from cathode to anode at pH 8.00
- mass to charge ratio of DNA is 1:1
- with few exceptions, DNA migrates through an electrical field based on its molecular weight
term used to describe having both positive and negative charges
ampholyte
- proteins; NOTE: @ the isoelectric point, pI, a
protein molecule has no net charge. At a pH above the protein’s pI, the protein is negatively charged and will migrate toward the anode during electrophoresis
describe serum protein electrophoresis
carried out at an alkaline pH above the pI of the serum protein = all proteins are negatively charged, but their mass to charge ratio varies
five zones of serum proteins in SPE
albumin (fastest)
alpha1
alpha2
beta proteins
gamma
What happens after SPE?
the proteins are fixed in the agarose and stained
Commonly used dyes for SPE
Coomassie Brilliant Blue, Amido Black and Ponceau S
What happens after staining SPE gels?
five zones are quantitated by densitometry
- visualization of nucleic acids in agarose gels
carried out by staining with a fluorescent dye. - gels photographed using a UV light source.
Agarose gels NOTused to quantitate nucleic acids
unequal migration across the well
dirty electrodes
uneven wetting of the gel
distorted protein zones
Bent applicator
Bubble introduced during sample application
Too much sample applied
unusual bands
Hemolyzed sample – increased B zone
Plasma sample – increase at B and y zone interface
Medication – unusual migration of albumin
how is capillary electrophoresis carried out?
in narrow, less than 500 um, fused silica capillary tubes up to 20 m in length
capillaries are reinforced with an exterior coating of polyimide
there’s a gap in the coating at the cathodic end of the capillary to allow for analyte detection
occurs at high-voltage, approximately 25 to 30 kV, the narrow glass tubes efficiently
dissipated heat
sample volume in capillary electrophoresis
1 to 50 nL
necessitates automated sample
injection
electrokinetic injection = a high voltage is applied to the sample, and kinetic energy drives the
sample into the capillary
hydrodynamic injection = one end of the capillary is placed in the sample, and
differential pressure is applied
T or F. In capillary serum protein electrophoresis, the proteins electrophoresis freely in an alkaline solution
T!
describe capillary serum protein electrophoresis
the negatively charged silanol groups of the fused silica capillary attract counterions from the buffer resulting in a strong electroendosmotic force moving the opposite direction to the electrical field
Where are samples injected for capillary SPE?
they are injected at the
anode (+), and despite their negative charge at an alkaline pH, the proteins migrate towards the cathode (-)
- the proteins are separated based on their electrophoretic mobility and endosmosis
where are proteins measured in capillary serum protein electrophoresis?
at the cathodic end of the capillary as they pass-through direct spectrophotometry at 200 to
225 nm
six distinct protein zones are recognized in capillary electrophoresis: albumin, alpha1, alpha2, beta1, beta2 and gamma
In DNA capillary electrophoresis, the DNA must be labelled with these before separation
labelled with fluorescent labels nucleotides before separation
what happens in capillary DNA electrophoresis?
DNA fragments are injected into the capillary by electrokinetic injection
DNA fragments electrophoresis through a flowable polymer of polyacrylamide at an alkaline pH
DNA capillary gel electrophoresis = interior of the capillary is coated to eliminate electroendosmotic flow; enhances separation efficiency
since all nucleic acids have the same mass-to-charge ratio, the flowable polymer acts as a …
molecular sieve to separate the nucleic acid based on length
- nucleic acids are injected at cathode (-) and migrates toward anode (+)
labelled nucleic acids are measured at ANODE end of capillary as they pass by a fluorescent light source and a detector
