ELECTROPHORESIS

Question 1

Define electrophoresis

Answer 1

Movement of charged particles by an external electrical field
- rate of migration is dependent on properties of support media, the electric field strength, and temperature

Question 2

Explain the principle of gel electrophoresis

Answer 2

• agarose gels have pores through which the analyte will migrate; determined by agarose concentration; separation by size is called molecular sieving
• DNA migration is generally by size (molecular sieving) since all DNA molecules have the same mass: charge ratio
• in protein electrophoresis, proteins separate into bands based on mass:charge ratios

Question 3

List 4 characteristics of capillary electrophoresis

Answer 3

• fused silica capillary tubes with exterior coating of polyimide
• high voltage (~25 to 30 kV)
• narrow tube efficiently dissipates heat
• uses electrokinetic or hydrodynamic injection

Question 4

Describe capillary Serum Protein Electrophoresis (SPE)

Answer 4

• no supporting medium (capillary zone electrophoresis)
• negatively charged silanol groups of silica capillary induces electroendosmosis force, moving sample in the opposite direction to the electrical field
• samples injected at anode (+); despite negative charge, proteins migrate towards cathode (-)
• proteins separate based on electrophoretic mobility and endosmosis

Question 5

Why is agarose gel more advantageous than starch or cellulose acetate ?

Answer 5

• no charge; minimal Electroendosmosis
• low affinity for proteins
• relatively clear when dried

Question 6

What is Electroendosmosis ?

Answer 6

• charged components of agarose gel (typically negative) attract counterions (cations) from buffer
• when the electric field is applied, cations will move towards the cathode, taking water with it (as well as all negative ions trapped within)

Question 7

Both protein and DNA are carried out at an __ pH

Answer 7

Both protein and DNA are carried out at an alkaline pH

Question 8

Under fixed current conditions, the rate of migration in a system __ as the ionic strength of the buffer increases

Answer 8

Under fixed current conditions, the rate of migration in a system decreases as the ionic strength of the buffer increases

Question 9

How does pH affect electrophoresis ?

Answer 9

Determines charge of analyte, and therefore its mobility

Question 10

How does ionic strength affect electrophoresis ?

Answer 10

• alters voltage (when current is fixed)
• increased ionic strength reduces rate of migration
• increased ionic strength = increased heat

Question 11

How might current negatively affect electrophoresis ?

Answer 11

too much current = excessive heat

Question 12

How does voltage affect electrophoresis ?

Answer 12

migration rate is proportional to voltage

Question 13

What is the difference between voltage and current ?

Answer 13

Voltage is the potential difference between two points in an electric field, which causes current/ electrons to flow in the circuit

Question 14

How does temperature affect electrophoresis ?

Answer 14

• unstable temperatures cause curved bands
• excess heat denatures proteins
• lower temperatures decrease migration rate

Question 15

How does time affect electrophoresis ?

Answer 15

Resolution of bands increases with time

Question 16

How does the support media affect electrophoresis ?

Answer 16

Electroendosmosis and pore size affect migration rate

Question 17

The rate of migration of an analyte in agarose gel electrophoresis is based on its __, __, and __.

Answer 17

The rate of migration of an analyte in agarose gel electrophoresis is based on its charge, molecular weight, and shape.

NOTE: if pore size of agarose is not sufficient for molecular sieving; proteins and nucleic acids migrate based on mass to charge ratio

Question 18

What is an ampholyte ? Give an example.

Answer 18

A species that has both positive and negative charges ie. proteins

Question 19

DNA has a __ charge due to its __.

Answer 19

DNA has a NEGATIVE charge due to its PHOSPHATE backbone.

Question 20

DNA migrates through an electric field from the __ (-/+) to the __ (-/+) at pH 8.

Answer 20

DNA migrates through an electric field from the CATHODE (-) to the ANODE (+) at pH 8.

Question 21

DNA migrate based on its __.

Answer 21

DNA migrate based on its molecular weight/ length/ size.

Question 22

At the isoelectric point (pI), protein has __ net charge.
At pH > pI, protein has __ net charge.
At pH < pI, protein has __ net charge.

What pH is SPE done on ?

Answer 22

At pI = no net charge
pH > pI; negative charge
pH < pI; positive charge

SPE is done at pH 8; proteins are negatively charged

Question 23

Although all proteins are __ charged in SPE, their __ varies.

Answer 23

Although all proteins are negatively charged in SPE, their mass/charge ratio varies.

Question 24

List 3 dyes used for SPE

Answer 24

1. Coomassie Brilliant Blue
2. Amino Black
3. Ponceau

Question 25

Five zones in SPE are quantitated by __.

Answer 25

Five zones in SPE are quantitated by densitometry.

Question 26

Causes of migration across the well

Answer 26

- dirty electrodes - uneven wetting of gel

Question 27

Causes of distorted protein zones

Answer 27

- bent applicator - bubble introduced during sample application - too much sample added

Question 28

Causes of unusual bands

Answer 28

- hemolyzed sample - plasma sample - medication

Question 29

Describe capillary DNA electrophoresis

Answer 29

- DNA must be labelled with fluorescent labelled-nucleotides before separation - sample is injected by electrokinetic injection at the cathode (-) and migrate to anode (+) - fragments migrate through polymer of polyacrylamide at alkaline pH - coated capillary (supporting medium) eliminates electroendosmosis and enhances separation efficiency - DNA migrates based on nucleic-acid length/ size

Question 30

Since all nucleic acids have the same __, the flowable polymer acts as a __ to separate the nucleic acids based on __.

Answer 30

Since all nucleic acids have the same MASS:CHARGE ratio, the flowable polymer acts as a molecular sieve to separate the nucleic acids based on MOLECULAR WEIGHT/LENGTH/SIZE.

Question 31

Which of the following technique separates proteins by electrophoresis and identifies the presence of a target protein with a labeled antibody ? a. Northern blotting b. Immunofixation c. Western blotting d. Immunoelectrophoresis

Answer 31

Western blotting

Question 32

Which of the following stains is suitable for CSF protein electrophoresis? a. Coomaisse brillant blue b. Silver nitrate c. Ethidium bromide d. Oil red O

Answer 32

b. Silver nitrate

Question 33

Which of the following support media provides the best resolution for nucleic acids? a. agar b. polyacrylamide c. agarose d. cellulose

Answer 33

b. polyacrylamide

Question 34

Which of the following describes conditioning of an electrophoresis capillary? Answer is in the textbook . a. flushing the capillary with 3 to 5 volumes of dilute NaOH b. flushing the capillary with 3 to 5 volumes of silyalation reagent c. flushing the capillary with 5 to 8 volumes of separation buffer d. flushing the capillary with 5 to 8 volumes of dilute HCl

Answer 34

c. flushing the capillary with 5 to 8 volumes of separation buffer

Question 35

What is the buffer of choice for Capillary Electrophoresis at pH 8? a. Tricine b. Borate c. Phosphate d. Tris

Answer 35

b. Borate

Question 36

T or F: In electrophoresis, as the temperature decreases so does the resistance.

Answer 36

FALSE; In electrophoresis, temperature and resistance are INVERSELY PROPRTIONAL. - hot things move faster

Question 37

T or F: In gel electrophoresis, resolution of molecules depends on the pore size of the media.

Answer 37

TRUE; In gel electrophoresis, resolution of molecules depends on the pore size of the media. - When pore size of gel matrix ≈ diameter of macromolecule, molecular sieving is possible and can enhance resolution

Question 38

Which of the following is an advantage of capillary columns? a. decreased back pressure b. increased sample capacity c. increased number of theoretical plates per column length d. decreased need for derivatization

Answer 38

c. increased number of theoretical plates per column length - Column efficiency = increased number of theoretical plates (N) per column length