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MLSCI360 > ELECTROPHORESIS > Flashcards

ELECTROPHORESIS Flashcards

1
Q

Define electrophoresis

A

Movement of charged particles by an external electrical field
- rate of migration is dependent on properties of support media, the electric field strength, and temperature

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2
Q

Explain the principle of gel electrophoresis

A
  • agarose gels have pores through which the analyte will migrate; determined by agarose concentration; separation by size is called molecular sieving
  • DNA migration is generally by size (molecular sieving) since all DNA molecules have the same mass: charge ratio
  • in protein electrophoresis, proteins separate into bands based on mass:charge ratios
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3
Q

List 4 characteristics of capillary electrophoresis

A
  • fused silica capillary tubes with exterior coating of polyimide
  • high voltage (~25 to 30 kV)
  • narrow tube efficiently dissipates heat
  • uses electrokinetic or hydrodynamic injection
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4
Q

Describe capillary Serum Protein Electrophoresis (SPE)

A
  • no supporting medium (capillary zone electrophoresis)
  • negatively charged silanol groups of silica capillary induces electroendosmosis force, moving sample in the opposite direction to the electrical field
  • samples injected at anode (+); despite negative charge, proteins migrate towards cathode (-)
  • proteins separate based on electrophoretic mobility and endosmosis
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5
Q

Why is agarose gel more advantageous than starch or cellulose acetate ?

A
  • no charge; minimal Electroendosmosis
  • low affinity for proteins
  • relatively clear when dried
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6
Q

What is Electroendosmosis ?

A
  • charged components of agarose gel (typically negative) attract counterions (cations) from buffer
  • when the electric field is applied, cations will move towards the cathode, taking water with it (as well as all negative ions trapped within)
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7
Q

Both protein and DNA are carried out at an __ pH

A

Both protein and DNA are carried out at an alkaline pH

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8
Q

Under fixed current conditions, the rate of migration in a system __ as the ionic strength of the buffer increases

A

Under fixed current conditions, the rate of migration in a system decreases as the ionic strength of the buffer increases

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9
Q

How does pH affect electrophoresis ?

A

Determines charge of analyte, and therefore its mobility

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10
Q

How does ionic strength affect electrophoresis ?

A
  • alters voltage (when current is fixed)
  • increased ionic strength reduces rate of migration
  • increased ionic strength = increased heat
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11
Q

How might current negatively affect electrophoresis ?

A

too much current = excessive heat

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12
Q

How does voltage affect electrophoresis ?

A

migration rate is proportional to voltage

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13
Q

What is the difference between voltage and current ?

A

Voltage is the potential difference between two points in an electric field, which causes current/ electrons to flow in the circuit

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14
Q

How does temperature affect electrophoresis ?

A
  • unstable temperatures cause curved bands
  • excess heat denatures proteins
  • lower temperatures decrease migration rate
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15
Q

How does time affect electrophoresis ?

A

Resolution of bands increases with time

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16
Q

How does the support media affect electrophoresis ?

A

Electroendosmosis and pore size affect migration rate

17
Q

The rate of migration of an analyte in agarose gel electrophoresis is based on its __, __, and __.

A

The rate of migration of an analyte in agarose gel electrophoresis is based on its charge, molecular weight, and shape.

NOTE: if pore size of agarose is not sufficient for molecular sieving; proteins and nucleic acids migrate based on mass to charge ratio

18
Q

What is an ampholyte ? Give an example.

A

A species that has both positive and negative charges ie. proteins

19
Q

DNA has a __ charge due to its __.

A

DNA has a NEGATIVE charge due to its PHOSPHATE backbone.

20
Q

DNA migrates through an electric field from the __ (-/+) to the __ (-/+) at pH 8.

A

DNA migrates through an electric field from the CATHODE (-) to the ANODE (+) at pH 8.

21
Q

DNA migrate based on its __.

A

DNA migrate based on its molecular weight/ length/ size.

22
Q

At the isoelectric point (pI), protein has __ net charge.
At pH > pI, protein has __ net charge.
At pH < pI, protein has __ net charge.

What pH is SPE done on ?

A

At pI = no net charge
pH > pI; negative charge
pH < pI; positive charge

SPE is done at pH 8; proteins are negatively charged

23
Q

Although all proteins are __ charged in SPE, their __ varies.

A

Although all proteins are negatively charged in SPE, their mass/charge ratio varies.

24
Q

List 3 dyes used for SPE

A
  1. Coomassie Brilliant Blue
  2. Amino Black
  3. Ponceau
25
Q

Five zones in SPE are quantitated by __.

A

Five zones in SPE are quantitated by densitometry.

26
Q

Causes of migration across the well

A
  • dirty electrodes
  • uneven wetting of gel
27
Q

Causes of distorted protein zones

A
  • bent applicator
  • bubble introduced during sample application
  • too much sample added
28
Q

Causes of unusual bands

A
  • hemolyzed sample
  • plasma sample
  • medication
29
Q

Describe capillary DNA electrophoresis

A
  • DNA must be labelled with fluorescent labelled-nucleotides before separation
  • sample is injected by electrokinetic injection at the cathode (-) and migrate to anode (+)
  • fragments migrate through polymer of polyacrylamide at alkaline pH
  • coated capillary (supporting medium) eliminates electroendosmosis and enhances separation efficiency
  • DNA migrates based on nucleic-acid length/ size
30
Q

Since all nucleic acids have the same __, the flowable polymer acts as a __ to separate the nucleic acids based on __.

A

Since all nucleic acids have the same MASS:CHARGE ratio, the flowable polymer acts as a molecular sieve to separate the nucleic acids based on MOLECULAR WEIGHT/LENGTH/SIZE.

31
Q

Which of the following technique separates proteins by electrophoresis and identifies the presence of a target protein with a labeled antibody ?

a. Northern blotting
b. Immunofixation
c. Western blotting
d. Immunoelectrophoresis

A

Western blotting

32
Q

Which of the following stains is suitable for CSF protein electrophoresis?

a.
Coomaisse brillant blue

b.
Silver nitrate

c.
Ethidium bromide

d.
Oil red O

A

b.
Silver nitrate

33
Q

Which of the following support media provides the best resolution for nucleic acids?

a.
agar

b.
polyacrylamide

c.
agarose

d.
cellulose

A

b.
polyacrylamide

34
Q

Which of the following describes conditioning of an electrophoresis capillary?

Answer is in the textbook .

a.
flushing the capillary with 3 to 5 volumes of dilute NaOH

b.
flushing the capillary with 3 to 5 volumes of silyalation reagent

c.
flushing the capillary with 5 to 8 volumes of separation buffer

d.
flushing the capillary with 5 to 8 volumes of dilute HCl

A

c.
flushing the capillary with 5 to 8 volumes of separation buffer

35
Q

What is the buffer of choice for Capillary Electrophoresis at pH 8?

a.
Tricine

b.
Borate

c.
Phosphate

d.
Tris

A

b.
Borate

36
Q

T or F: In electrophoresis, as the temperature decreases so does the resistance.

A

FALSE; In electrophoresis, temperature and resistance are INVERSELY PROPRTIONAL.

  • hot things move faster
37
Q

T or F: In gel electrophoresis, resolution of molecules depends on the pore size of the media.

A

TRUE; In gel electrophoresis, resolution of molecules depends on the pore size of the media.

  • When pore size of gel matrix ≈ diameter of macromolecule, molecular sieving is possible and can enhance resolution
38
Q

Which of the following is an advantage of capillary columns?

a.
decreased back pressure

b.
increased sample capacity

c.
increased number of theoretical plates per column length

d.
decreased need for derivatization

A

c.
increased number of theoretical plates per column length

  • Column efficiency = increased number of theoretical plates (N) per column length

MLSCI360 (22 decks)

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