360 - Electrophoresis Flashcards by Richen Basig

The rate of
migration of an analyte during electrophoresis is dependent on the properties of …

the support media,
the electrical field strength,
the temperature

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T or F. DNA migration through agarose is considered a function of size, as all molecules have the same mass to charge ratio

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proteins and nucleic acids migrate through agarose gels based on their …

mass to charge ratio

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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

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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

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This determines the net charge of the analytes during electrophoresis

the pH ofthe buffer

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This determines the net charge of the analytes during electrophoresis

the pH of the buffer

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T or F. Both protein and DNA electrophoresis are carried out at an acidic pH

F! ALKALINE

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____ in the buffer carry the current

Ions

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Under fixed current conditions, the rate of migration of macromolecules in a system _______________ as the ionic strength (conductivity) of the buffer increases

decreases

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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

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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

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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

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how does pH affect electrophoresis?

determines the charge of analyte and therefore it’s mobility

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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

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how does current affect electrophoresis?

too much current results in excessive heat production

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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

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

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