Module 11

Question 1

What is zone electrophoresis?

Answer 1

Separation of molecules based on size and charge on a porous support medium.

Bands of particles are visualized and quantified.

Question 2

What is migration?

Answer 2

The distance molecules move from the point of application.

Question 3

What is migration rate dependent on?

Answer 3

Net charge of molecule

Size and shape of molecule

Electric field strength

Support medium

Temperature

Question 4

What is mobility?

Answer 4

The velocity that a particle moves with through a support medium in an electric field.

Question 5

What is mobility proportional to?

Answer 5

Directly- net charge

Indirectly- size

Question 6

What are the components of an electrophoretic system?

Answer 6

Support medium

Buffer

Chamber with electrodes (anode and cathode)

Power supply- provides current

Question 7

What are the functions of buffer?

Answer 7

Fix pH of process

Carry electric current

Question 8

What is the process of electrophoresis?

Answer 8

Sample is applied to gel

Gel is placed in chamber with buffer

Current is applied

Proteins migrate for a specified time

Gel is dried or fixed and then stained

Question 9

What factors affect separation?

Answer 9

pH

Ionic strength

Voltage and current

Support media

Question 10

How does pH affect separation?

Answer 10

Net protein charge is dependent of pH.

Acid- amino group is positively charged, migrated to cathode

Base- carboxyl group is negatively charged migrates to anode.

Question 11

What is the isoelectric point and how does it affect separation?

Answer 11

pH at which the net charge of protein is zero.

Difference in buffer pH relative to pI of protein determines direction and magnitude.

pH>pI- positive charge, to cathode

pH

Question 12

What is the normal movement of proteins?

Answer 12

Buffer pH~8.6

Protein pH~4.9-5.9

Therefore negatively charged, move to anode.

Question 13

How does ionic strength affect separation?

Answer 13

Proteins collect clouds of electrolyte ions from the buffer.

Affects migration.

Higher- low mobility, small migration, high heat production (increased wick flow), high resolution.

Lower- high mobility, large migration, low heat production, low resolution

Question 14

What buffers are typically used?

Answer 14

Barbital

Tris-boric acid EDTA

Question 15

How does voltage and current affect separation?

Answer 15

Application of voltage produces an electric field.

Resistance is the opposition of current trying to flow.

Heat is produced by the movement of electrons against resistance.

Heat reduces resistance.

V = IR

Question 16

How can voltage and current be altered to address heat production?

Answer 16

Constant voltage- as resistance decreases, current must increase to maintain voltage, faster migration rate

Constant current- resistance decreases, voltage must decrease to maintain current, no change in migration rate

Question 17

Why is agarose gel a good support medium?

Answer 17

Low affinity for proteins.

Free of ionizable groups.

Naturally clear after drying.

Question 18

What are limitations/sources of problems with electrophoresis?

Answer 18

Wick flow

Electroendosmosis

Question 19

What is wick flow?

Answer 19

Heat produced causes solvent evaporation.

Draws buffer into support from both ends.

Buffer flow affects rate and length of migration.

Question 20

How is wick flow avoided?

Answer 20

Ensure lid is closed

Cooling the support

Question 21

What is electroendosmosis?

Answer 21

Chemical groups in the support absorb OH ions giving it a negative charge.

Positive ions cluster around the negative charges.

When current is applied the positive clouds move to the cathode causing solvent to flow with it.

Macromolecules may be forced to remain immobile or swept towards the cathode.

Question 22

How can electroendosmosis be limited?

Answer 22

Using media with minimal surface changes or ionizable groups.

Question 23

What is the purpose of staining?

Answer 23

Used to visualize and identify the different bands/zones.

Question 24

What dyes are proteins stained with?

Answer 24

Coomassie blue

Amido black

Question 25

How are the bands/zones quantified?

Answer 25

Densitometer (photometer) scans the support medium.

Absorbance of each band is measured and displayed as a series of peaks.

Area under each peak is proportional to the concentration of the sample fraction in that band.

Question 26

What is the order of protein migration (fastest to slowest)?

Answer 26

Albumin

a1 globulin

a2 globulin

B1 and 2 globulin

Gamma globulin

Question 27

How are gels used for diagnosis?

Answer 27

Look for increases/decreases in relative band quantities or the absence or presence of bands.

Question 28

What are some characteristic patterns of disease?

Answer 28

Decreases in albumin- liver disease and renal disorders

Cirrhosis and inflammation- characteristic pattern

Increased gamma region- infections

M peaks- monoclonal antibodies indicative of monoclonal gammopathy (eg. multiple myeloma), followed up with IFE

Question 29

What are some sources of error and their characteristics?

Answer 29

No power- no migration

High voltage- fast migration, indistinct bands

Wrong time- pattern long or short

Gel not secure- no migration or uneven pattern

No buffer- no migration

Incorrect buffer- unpredictable pattern (depends on pH)

Bubbles- uneven sample application

Gel not dry prior to staining- whatever is wet will stain

Gel damage- uneven pattern

Undiluted serum samples- dark pattern, bands hard to distinguish

Not fixed- very pale

Question 30

What is electrophoresis?

Answer 30

The migration of charged particles in an electric field.