Principles of Electrophoresis

Question 1

What is the principle of electrophoresis

Answer 1

It’s a separation technique based on the migration of charged solutes/particles in a liquid medium under influence of an electrical field

Question 2

Electrophoretic mobility

Answer 2

Dissimilar molecules in a mixture will separate as they move at different rates - size influences, bigger molecules will move slower

Question 3

What can electrophoresis be used to isolate & analyse

Answer 3

• Proteins
• Amino acids
• Nucleic acids

Question 4

What is generated

Answer 4

An electrophoretogram

Question 5

What are zones visualised with

Answer 5

An analyte specific stain - e.g: Sybr Green

Question 6

Function of the electrodes

Answer 6

Constant voltage/current between the 2 electrodes creates an electric field that causes movement of charged ions

Question 7

Anode

Answer 7

Pos electrode that attracts neg charged anions

Question 8

Cathode

Answer 8

Neg electrode that attracts pos charged cations

Question 9

Function of the support medium

Answer 9

Provides the connection between the 2 electrodes

Question 10

What does the electrophoretic mobility depend on

Answer 10

1. Physical characteristics
2. Method of electrophoresis

Question 11

3 types of support media

Answer 11

1. Porous insoluble gels (gel electrophoresis)
2. Inert membranes (cellulose acetate)
3. Pure buffer solutions (capillary electrophoresis)

Question 12

What is agarose

Answer 12

Linear polysaccharide polymer made of repeating units of agarobiose

Question 13

Pro of agarose gel

Answer 13

Large range of separation

Question 14

Con of agarose gel

Answer 14

Low resolving power

Question 15

What is polyacrylamide & how is it prepared

Answer 15

A polymer, prepared by heating acrylamide with a catalyst, with or without crosslinking agents

Question 16

3 characteristics of polyacrylamide gels

Answer 16

1. thermostable
2. transparent
3. relatively chemically inert

Question 17

What are analytes separated based on with polyacrylamide gel

Answer 17

Based on charge

Question 18

Pro & Con of polyacrylamide gel

Answer 18

Pro: High resolving power (can resolve DNA that differs by as little as 2% in length)
Con: Low range of separation

Question 19

How are the inert membranes, cellulose acetate, made

Answer 19

Made by treating cellulose with acetic anhydride

Question 20

What do the cellulose acetate membranes need to be soaked in before use

Answer 20

Buffer

Question 21

What is separation based on in capillary electrophoresis

Answer 21

Separation based on size to charge ratio

Question 22

in capillary electrophoresis, what direction do all ions move in & what causes this

Answer 22

In the same direction by electro-osmotic flow

Question 23

Advantages of capillary electrophoresis

Answer 23

1. Enhanced separation efficiency
2. Reduced separation time
3. Lower handling errors
4. Small sample volumes analysed

Question 24

System components of capillary electrophoresis

Answer 24

1. Power supply
2. Buffers

Question 25

What does high voltage improve in capillary electrophoresis

Answer 25

Improves resolution & decreases time needed for separation

Question 26

Function of buffers in capillary electrophoresis

Answer 26

Carries the applied current & establishes the pH

Question 27

What do high ionic strength buffers yield? what do these produce and then affect/

Answer 27

High ionic strength buffers yield sharp bands. But these produce more Joule heat. This can affect heat labile proteins.

Question 28

What are the factors affecting migration rates of molecules

Answer 28

1. Molecular weight, size & shape 2. Buffer pH 3. Supporting media 4. Temp 5. Electrical voltage 6. Migration time

Question 29

What are the 3 major variations possible of conventional electrophoresis (agarose gel electrophoresis)

Answer 29

1. Isoelectric focusing (IEF) 2. Rocket immunoelectrophoresis (RIE) 3. SDS-Polyacrylamide gel electrophoresis (PAGE)

Question 30

What is used to separate bands in Isoelectric Focusing and what is the separation based on

Answer 30

Isoelectric focusing uses a pH gradient to separate, based on isoelectric point

Question 31

In isoelectric focusing, how is gradient formed

Answer 31

Gradient is formed using low molecular weight ampholytes with a range of pI values

Question 32

In isoelectric focusing, what do molecules in a mixture migrate until

Answer 32

The molecules in the mixture migrate until their isoelectric point matches the local pH (net charge is then 0)

Question 33

Principle of immunoelectrophoresis

Answer 33

Use of antibody impregnated agarose gel at high pH to quantitate proteins

Question 34

How does rocket immunoelectrophoresis work

Answer 34

1. Samples applied at cathode, where antigen is in excess 2. As proteins migrate, the concentration decreases 3. As large stable immune complexes form, these precipitate & can no longer migrate 4. Sharp arc of precipitated antigen-antibody complexes that resemble rocket 5. Height of precipitation arc is proportional to con of protein in sample

Question 35

What is the height of the precipitation arc proportional to

Answer 35

Height of precipitation arc is proportional to concentration of protein in sample

Question 36

What is the difference in dissociating vs non-dissociating PAGE (polyacrylamide gel electrophoresis)

Answer 36

With (SDS-PAGE) or without (nondenaturing PAGE, no SDS) SDS = Sodium Dodecyl Sulfate

Question 36

What is the difference between continuous & discontinuous PAGE

Answer 36

* Continuous: uses a single gel that acts as a molecular sieve with the same buffer ions in sample and gel5 * Discontinuous: uses 2 gel systems-> Stacking (large pores) and resolving (small pores) with different buffers in gel compared to buffer reservoir

Question 37

Clinical application of serum protein electrophoresis

Answer 37

Separates serum proteins into 5 major bands - albumin, alpha 1 globulins, alpha 2 globulins , beta globulins & gamma globulins

Question 38

What type of data do electrophoretograms give

Answer 38

Qualitative & quantitative

Question 39

In serum protein electrophoresis, which protein band is increased in multiple myeloma

Answer 39

The gamma band

Question 40

Principle of Hb electrophoresis

Answer 40

Haemolysed blood sample analysed & Hb content separated Separation of normal proteins & detection of Hb variants e

Question 41

Examples of isoenzymes that can be separated & quantitated electrophoretically

Answer 41

Creatine kinase (CK) & lactate dehydrogenase (LD)

Question 42

Separation of isoenzymes steps

Answer 42

1. After separation, substrates added 2. Rxn occurs leading to product that is detected

Question 43

What can isoenzyme separation provide info on

Answer 43

Organ damage - different isoenzymes produced by different organs