Electrophoresis

Question 1

What is electrophoresis

Answer 1

the movement of charge particles by an external electrical field

Question 2

What is the rate of migration during electrophoresis dependant on

Answer 2

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

Question 3

What does electrophoresis do

Answer 3

separate and analyze proteins and nucleic acids

Question 4

What is agarose

Answer 4

a support medium made of purified agar

Question 5

How is the pore size of agarose gel controlled

Answer 5

by concentration

Question 6

How do proteins separate in electrophoresis

Answer 6

they separate into bands based on their mass to charge ratio

Question 7

How does DNA usually migrate through an agarose gel

Answer 7

based on size since they all have the same charge

Question 8

Why is agarose preferred to other methods

Answer 8

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

Question 9

What is electroendosmosis

Answer 9

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

Question 10

What does the pH of a buffer do

Answer 10

determines the net charge of the analytes during electrophoresis

Question 11

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

Answer 11

it decreases

Question 12

How do we keep the current constant

Answer 12

we decrease the voltage which decreases the applied electrical field

Question 13

How do we maintain constant voltage

Answer 13

increase the current as the ionic strength of the buffer increases

Question 14

What is the risk of a high current

Answer 14

overheating and convection currents

Question 15

What effect does pH have on electrophoresis

Answer 15

determines the charge of analyte and therefore its mobility

Question 16

What effect does ionic strength have on electrophoresis

Answer 16

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

Question 17

What effect does current have on electrophoresis

Answer 17

too much current results in excessive heat production

Question 18

What effect does voltage have on electrophoresis

Answer 18

migration rate is proportional to voltage

Question 19

What effect does temperature have on electrophoresis

Answer 19

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

Question 20

What effect does time have on electrophoresis

Answer 20

resolution of bands increases with time

Question 21

What effect does support media have on electrophoresis

Answer 21

electroendosmosis and pore size effect migration rates

Question 22

How does DNA migrate in electrophoresis

Answer 22

from the cathode to the anode

Question 23

What is an ampholyte

Answer 23

something that has both a positive and negative charge

Question 24

What effect does the isoelectric point have on a protein

Answer 24

it causes it to have no net charge

Question 25

How does pH above the isoelectric point do to a protein

Answer 25

causes it to be negatively charged

Question 26

How does pH below the isoelectric point do to a protein

Answer 26

causes it to be positively charged

Question 27

What are the conditions for serum protein electrophoresis

Answer 27

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

Question 28

What occurs after serum protein electrophoresis

Answer 28

proteins are fixed in the agarose and stained

Question 29

What are common dyes for SPE

Answer 29

coomassie brilliant blue, amido black and ponceau S

Question 30

What causes unequal migration across the well

Answer 30

dirty electrodes, uneven wetting of the gel

Question 31

What causes distorted protein zones

Answer 31

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

Question 32

What causes usual bands

Answer 32

hemolyzed sample, plasma sample, medication

Question 33

How is capillary electrophoresis carried out

Answer 33

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

Question 34

How are capillaries reinforced

Answer 34

exterior coating of polyimide

Question 35

What kind of sample injection is used for capillary electrophoresis

Answer 35

hydrodynamic injection

Question 36

How does hydrodynamic injection work

Answer 36

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

Question 37

How do proteins move in capillary electrophoresis

Answer 37

freely in alkaline solution without supporting medium

Question 38

What causes an electroendosmosis force in capillary electrophoresis

Answer 38

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

Question 39

What direction do proteins migrate in capillary electrophoresis

Answer 39

towards the cathode

Question 40

How are proteins separated in capillary electrophoresis

Answer 40

based on their electrophoretic mobility and endosmosis

Question 41

What is an important step for DNA capillary electrophoresis

Answer 41

DNA must be labelled with fluorescent labeled nucleotides before separation

Question 42

How does DNA capillary electrophoresis work

Answer 42

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

Question 43

How is DNA separated in capillary electrophoresis

Answer 43

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

Question 44

How does DNA move through capillary electrophoresis

Answer 44

injected at the cathode and moves to wards the anode