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Clinical Biochem > Electrophoresis > Flashcards

Electrophoresis Flashcards

1
Q

What is electrophoresis

A

the movement of charge particles by an external electrical field

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2
Q

What is the rate of migration during electrophoresis dependant on

A

the properties of the support media, electric field strength and temperature

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3
Q

What does electrophoresis do

A

separate and analyze proteins and nucleic acids

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4
Q

What is agarose

A

a support medium made of purified agar

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5
Q

How is the pore size of agarose gel controlled

A

by concentration

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6
Q

How do proteins separate in electrophoresis

A

they separate into bands based on their mass to charge ratio

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7
Q

How does DNA usually migrate through an agarose gel

A

based on size since they all have the same charge

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8
Q

Why is agarose preferred to other methods

A

it is easy to handle, has no charge, and contributes minimally to electroendosmosis

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9
Q

What is electroendosmosis

A

charged components of the support media attract counterions from the buffer which move towards the oppositely charged electrode taking the solvent with them, this forces the net movement of solvent in one direction which can slow down or reverse analyte migaration

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10
Q

What does the pH of a buffer do

A

determines the net charge of the analytes during electrophoresis

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11
Q

How does the rate of migration in a system change as conductivity of the buffer increases

A

it decreases

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12
Q

How do we keep the current constant

A

we decrease the voltage which decreases the applied electrical field

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13
Q

How do we maintain constant voltage

A

increase the current as the ionic strength of the buffer increases

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14
Q

What is the risk of a high current

A

overheating and convection currents

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15
Q

What effect does pH have on electrophoresis

A

determines the charge of analyte and therefore its mobility

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16
Q

What effect does ionic strength have on electrophoresis

A

alters voltage, increased ionic strength usually reduces migration rate, increased ionic strength usually increases heating

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17
Q

What effect does current have on electrophoresis

A

too much current results in excessive heat production

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18
Q

What effect does voltage have on electrophoresis

A

migration rate is proportional to voltage

19
Q

What effect does temperature have on electrophoresis

A

temperature gradients cause curved bands, excess heat can denature proteins, lower temperatures decrease migration rates

20
Q

What effect does time have on electrophoresis

A

resolution of bands increases with time

21
Q

What effect does support media have on electrophoresis

A

electroendosmosis and pore size effect migration rates

22
Q

How does DNA migrate in electrophoresis

A

from the cathode to the anode

23
Q

What is an ampholyte

A

something that has both a positive and negative charge

24
Q

What effect does the isoelectric point have on a protein

A

it causes it to have no net charge

25
Q

How does pH above the isoelectric point do to a protein

A

causes it to be negatively charged

26
Q

How does pH below the isoelectric point do to a protein

A

causes it to be positively charged

27
Q

What are the conditions for serum protein electrophoresis

A

alkaline pH above the isoelectric point of all proteins allowing them all to be negatively charged

28
Q

What occurs after serum protein electrophoresis

A

proteins are fixed in the agarose and stained

29
Q

What are common dyes for SPE

A

coomassie brilliant blue, amido black and ponceau S

30
Q

What causes unequal migration across the well

A

dirty electrodes, uneven wetting of the gel

31
Q

What causes distorted protein zones

A

bent applicator, bubble introduced during sample application, too much sample applied

32
Q

What causes usual bands

A

hemolyzed sample, plasma sample, medication

33
Q

How is capillary electrophoresis carried out

A

in narrow, less than 500nm fused silica capillary tubes up to twenty meters in length

34
Q

How are capillaries reinforced

A

exterior coating of polyimide

35
Q

What kind of sample injection is used for capillary electrophoresis

A

hydrodynamic injection

36
Q

How does hydrodynamic injection work

A

one end of the capillary is placed in the sample and differential pressure is applied

37
Q

How do proteins move in capillary electrophoresis

A

freely in alkaline solution without supporting medium

38
Q

What causes an electroendosmosis force in capillary electrophoresis

A

the negatively charged silanol groups of the fused silica capillary which attract counterions from the buffer

39
Q

What direction do proteins migrate in capillary electrophoresis

A

towards the cathode

40
Q

How are proteins separated in capillary electrophoresis

A

based on their electrophoretic mobility and endosmosis

41
Q

What is an important step for DNA capillary electrophoresis

A

DNA must be labelled with fluorescent labeled nucleotides before separation

42
Q

How does DNA capillary electrophoresis work

A

DNA fragments move through a flowable polymer of polyacrylamide at an alkaline pH, the interior of the capillary is coated to eliminate electroendosmosis

43
Q

How is DNA separated in capillary electrophoresis

A

the flowable polymer acts as a molecular sieve to separate the nucleic acids based on length

44
Q

How does DNA move through capillary electrophoresis

A

injected at the cathode and moves to wards the anode

Clinical Biochem (15 decks)

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