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MLSCI 262 > 360 - Electrophoresis > Flashcards

360 - Electrophoresis Flashcards

1
Q

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

A

the support media,
the electrical field strength,
the temperature

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2
Q

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

A

T!

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3
Q

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

A

mass to charge ratio

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4
Q

why is agarose preferable over starch and cellulose acetate gels?

A

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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5
Q

Describe electroendosmosis:

A
  • 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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6
Q

This determines the net charge of the analytes during electrophoresis

A

the pH ofthe buffer

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7
Q

This determines the net charge of the analytes during electrophoresis

A

the pH of the buffer

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8
Q

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

A

F! ALKALINE

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9
Q

____ in the buffer carry the current

A

Ions

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10
Q

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

A

decreases

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11
Q

how do we keep the system at a constant current?

A

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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12
Q

To maintain constant voltage conditions, the current must be _________ as the ionic strength (conductivity)
of the buffer __________

A

increased; increases

migration velocity of the macromolecules is unchanged BUT
too high of a current may cause overheating

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13
Q

what can excessive heat lead to?

A

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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14
Q

how does pH affect electrophoresis?

A

determines the charge of analyte and therefore it’s mobility

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15
Q

how does ionic strength affect electrophoresis?

A
  • 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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16
Q

how does current affect electrophoresis?

A

too much current results in excessive heat production

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17
Q

how does voltage affect electrophoresis?

A

migration rate is proportional to voltage

18
Q

how does temperature affect electrophoresis?

A

Temperature gradients cause curved bands

Excess heat can denature proteins

Lower temperatures decrease migration rates

19
Q

how does time affect electrophoresis?

A

band resolution increases with time

20
Q

how does the suppot media affect electrophoresis?

A

Electroendosmosis and pore size affect migration rates

21
Q

how does the support media affect electrophoresis?

A

Electroendosmosis and pore size affect migration rates

22
Q

The rate migration of an analyte in electrophoresis is based on the analyte’s…

A

charge, molecular weight, and shape

23
Q

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

A

T!

24
Q

DNA has a __________ charge

A

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
25
Q

term used to describe having both positive and negative charges

A

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
26
Q

describe serum protein electrophoresis

A

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

27
Q

five zones of serum proteins in SPE

A

albumin (fastest)
alpha1
alpha2
beta proteins
gamma

28
Q

What happens after SPE?

A

the proteins are fixed in the agarose and stained

29
Q

Commonly used dyes for SPE

A

Coomassie Brilliant Blue, Amido Black and Ponceau S

30
Q

What happens after staining SPE gels?

A

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
31
Q

unequal migration across the well

A

dirty electrodes

uneven wetting of the gel

32
Q

distorted protein zones

A

Bent applicator
Bubble introduced during sample application
Too much sample applied

33
Q

unusual bands

A

Hemolyzed sample – increased B zone
Plasma sample – increase at B and y zone interface
Medication – unusual migration of albumin

34
Q

how is capillary electrophoresis carried out?

A

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

35
Q

sample volume in capillary electrophoresis

A

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

36
Q

T or F. In capillary serum protein electrophoresis, the proteins electrophoresis freely in an alkaline solution

A

T!

37
Q

describe capillary serum protein electrophoresis

A

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

38
Q

Where are samples injected for capillary SPE?

A

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
39
Q

where are proteins measured in capillary serum protein electrophoresis?

A

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

40
Q

In DNA capillary electrophoresis, the DNA must be labelled with these before separation

A

labelled with fluorescent labels nucleotides before separation

41
Q

what happens in capillary DNA electrophoresis?

A

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

42
Q

since all nucleic acids have the same mass-to-charge ratio, the flowable polymer acts as a …

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

MLSCI 262 (21 decks)

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