Module 11: Electrophoresis

Question 1

What is electrophoresis

Answer 1

the migration of charged particles in an electric field

Question 2

Zone electrophoresis

Answer 2

typical for clinical labs

Results in a separation of particles based upon size and charge

Question 3

migration

Answer 3

the distance the molecules move from the point of application

Question 4

Things that affect rate of migration (5)

Answer 4

net electric charge of molecule
size and shape
electric field strength
support medium
temperature

Question 5

Electrophoretic mobility

Answer 5

the velocity that a particles moves through a support medium in an electric field
dependent on net charge and size

Question 6

Electrophoretic mobility is directly proportionate to

Answer 6

net charge

Question 7

Electrophoretic mobility is indirectly proportionate to

Answer 7

size

Question 8

Basic electrophoretic system consists of (4)

Answer 8

Support medium (gel such as agarose; sample applied to gel)
Buffer (electrolyte solution that fix pH of process and carry electric current)
Chamber with electrodes (filled with buff and gel placed inside; 2 electrodes - the anode (POS charged) and cathode (NEG charged)
Power supply (provide current to system for electrical field; current and voltage can be regulated)

Question 9

Basic electrophoresis process (5)

Answer 9

Sample applied to gel at “point of application”
Gel placed in chamber filled with buffer
Electric current applied by power supply
Proteins migrate in the electrical field; after specific amount of time, power is turned off and gel is removed
Gel is dried/fixed and then stained to visualize separated bands

Question 10

4 factors affecting separation

Answer 10

pH
Ionic Strength
Voltage and current
Support Media

Question 11

How pH affects separation

Answer 11

Proteins are amphoteric or ampholytes (can be pos/neg charged)
Net charge of protein depends on pH of solution they are in
In acid pH, amino group becomes pos charged
In basic pH, amino group becomes neg charged
pI is dependent upon proteins structure and amino acid composition
When protein is placed in pH specific buffer, difference between buffer pH and pI of protein determines magnitude and type of charge on protein

Question 12

In acid pH, proteins amino group becomes

Answer 12

positively charged

Question 13

In basic pH, proteins carboxyl group becomes

Answer 13

negatively charged

Question 14

Isoelectric point (pI)

Answer 14

the specific pH level that net charge on the protein is zero

Question 15

When a protein is placed in a buffer with a specified pH

Answer 15

the difference in the buffer pH relative to the isolelectric point of the protein determines the magnitude and type of charge on the protein

Question 16

If the pH of the buffer is the same as the pI, the net charge is

Answer 16

zero

Question 17

If the pH of the buffer is LESS than the pI, net charge is

Answer 17

positive

The protein is a cation and migrates towards the negatively charge CATHODE

Question 18

If the pH of the buffer is MORE than the pI, the net charge is

Answer 18

negative

The protein is an anion and migrates to the positively charged ANODE

Question 19

***IMPORTANT

For most EP methods, the buffer has an alkaline/basic pH, usually 8.6, so serum proteins will be

Answer 19

negatively charged anions and migrate towards the anode

Question 20

The further apart the buffer pH and pI,

Answer 20

the greater the net charge on the protein and therefore the greater its mobility

Question 21

the higher the concentration of electrolytes in a buffer,

Answer 21

the higher the Ionic strength

Question 22

As proteins move through the buffer, they collect “clouds” of

Answer 22

electrolyte ions from the buffer

Question 23

High ionic strength

Answer 23

Collects larger clouds of electrolyte ions from buffer
Low motility
Small total migration
Higher heat production (may denature proteins and increase wick flow)
**High resolution (each band is more distinct)

Question 24

Low ionic strength

Answer 24

Higher (faster) mobility
Larger total migration
lower heat production
lower resolution

Question 25

Voltage and current

Answer 25

Ohm's Law E=IxR Heat is produced due to movement of electrons against resistance BUT heat reduces the resistance (increases the conductance) by increasing rate of evaporation of water from support medium and increasing migration rate

Question 26

2 ways voltage and current can be applied to address heat production

Answer 26

1) Constant Voltage - as resistance decreases, current must increase to maintain constant voltage - change in current flow = faster migration rate (affects separation pattern) 2) Constant current! - as resistance decreases, voltage must decrease to maintain constant current - no change in current = no change to separation pattern

Question 27

With constant current, migration rate remains relatively

Answer 27

constant

Question 28

Support Media

Answer 28

Usually agarose gel - low affinity for proteins - free of ionizable groups (less interference during procedure) - Naturally clear after drying which provides for good quality densitometric scans

Question 29

2 limitations in electrophoresis

Answer 29

wick flow | electroendosmosis

Question 30

Wick flow

Answer 30

heat = evaporation of water from support medium this draws buffer into the support from both ends (anode and cathode) Flow of buffer from both directions affects rate and length of migration

Question 31

How can wick flow be avoided

Answer 31

ensuring lid of EP chamber is in place and support is cooled during high-voltage applications

Question 32

Electroendosmosis

Answer 32

In certain support media with chemical groups that absorb OH- ions that give the support media a net neg charge Neg charges fixed on support medium attract positive ions from buffer When current is applied, charges attached to support remain fixed but pos charged clouds of ions are free to move to cathode cause solvent to flow with it Macro molecules in solution can only move towards anode IF they sufficiently charged or large enough. Otherwise they may remain immobile or be swept backwards towards the cathode

Question 33

How can electroendosmosis be limited

Answer 33

by use of support media with minimal surface charges or ionizable groups

Question 34

Staining

Answer 34

used to visualize and identify the different bands | Coomassie blue or Amido black usually used

Question 35

SPE gel regions

Answer 35

``` Albumin a1 a2 Beta **Point of application** Gamma region (swept backwards by electroendosmosis) ```

Question 36

Quantitation (densitometry)

Answer 36

electrophoretic support medium is passed through optical beam of densitometer The absorbance of each fraction of a sample is measured and displayed on chart as peaks Some EP systems use digital scanners For a given peak, the area UNDER the peak is proportional to concentration of the sample fraction in that band

Question 37

Albumin

Answer 37

fasting moving protein and therefore moves farthest towards the anode during EP

Question 38

Globulins

Answer 38

alpha 1 alpha 2 beta gamma

Question 39

Cathodic migration

Answer 39

gamma globulins separates towards the cathode side of the point of application due to electroendosmosis

Question 40

EP gels are examined for

Answer 40

increases and decreases in relative quantities of each band and the presence or absence of bands

Question 41

liver disease and renal disorders show a decrease in

Answer 41

albumin

Question 42

infections show broad increases in

Answer 42

the gamma region

Question 43

M-peaks

Answer 43

separate distinct peaks in the gamma region may represent monoclonal antibodies may indicate multiple myeloma

Question 44

If an M-peak shows up, follow up with an

Answer 44

IFE test

Question 45

Error: incorrect buffer used

Answer 45

Appearance: unpredictable pattern | depends on pH of the buffer

Question 46

Error: Gel not completely dry prior to staining

Answer 46

wherever gel was wet, it is permanently stained, therefore unable to determine pattern