Module 11: Electrophoresis

Question 1

Principle of electrophoresis

Answer 1

Separation of proteins based on charge and size in an electrical field

Question 2

Electrophoresis clinical uses

Answer 2

Separate serum proteins to identify protein disorders (multiple myeloma: m-peak in gamma region; liver cirrhosis: characteristic beta-gamma bridging)

Question 3

Electrophoretic mobility

Answer 3

Velocity the protein migrates

Question 4

Factors that affect rate of migration

Answer 4

Net charge on the molecule
Size and shape of the molecule
Electric field strength
Properties of the support medium
Temperature of the operation

Question 5

Amphoteric

Answer 5

Can have either a positive or negative charge

Proteins are amphoteric depending on the pH of the solution they are in

Question 6

Isoelectric point (pI)

Answer 6

pH where the net charge of the protein is zero

Question 7

If pI > pH it will have a (pos/neg) charge and migrate towards the (anode/cathode)

Answer 7

Positive

Cathode

Question 8

If pI is less than pH it will have a (pos/neg) charge and migrate towards the (anode/cathode)

Answer 8

Negative

Anode

Question 9

In pH of 8.6, proteins are (anions/cations) and migrate towards the (anode/cathode)

Answer 9

Anions

Anode

Question 10

Buffers function to:

Answer 10

Maintain pH

Conduct electrical current

Question 11

Higher ionic strength buffers = (slower and shorter/faster and longer) overall migration and produce (better/worse) resolution

Answer 11

Slower and shorter

Better

Question 12

Benefits of agarose gel for electrophoresis

Answer 12

Few ionizable groups therefore less interference due to electroendosmosis
Low protein affinity

Question 13

Rate of migration is determined by the:

Answer 13

Current

Question 14

Ohm’s Law

Answer 14

E = I x R
Voltage = current (amps) x resistance (ohms)

Question 15

Effects of constant voltage

Answer 15

As heat is generated, resistance decreases, results in increase in current therefore migration increases

Question 16

Effects of constant current

Answer 16

As heat is generated, resistance decreases, voltage decreases, current remains constant, therefore migration is constant

Question 17

Densitometer

Answer 17

Uses photometry to measure absorbance of bands after gel is dried and stained

Question 18

Wick flow

Answer 18

Evaporation of buffer in the middle draws up more buffer from the ends of the gel
Controlled by keeping lid on system and cooling temperature

Question 19

Electroendosmosis

Answer 19

Flow of buffer ions and solvent in opposite direction of protein migration
Buffer cations and solvent flow toward cathode, gamma globulins are moved towards cathode (cathodic migration)

Question 20

How is electroendosmosis minimized?

Answer 20

Using media with few ionizable groups and low protein affinity

Question 21

Electrophoretic mobility is directly proportional to the _____ and inversely proportional to the ______ of the molecule.

Answer 21

Net charge

Size

Question 22

What is the advantage of using constant current over constant voltage?

Answer 22

Migration rate remains relatively constant

Question 23

Increasing ionic concentration of the buffer (increases/decreases) migration?

Answer 23

Decreases

Question 24

Increasing ionic concentration of the buffer (increases/decreases) resolution?

Answer 24

Increases

Question 25

Increasing ionic concentration of the buffer (increases/decreases) heat generation?

Answer 25

Increases

Question 26

Most plasma protein pIs:

Answer 26

4.9-5.9