Electrophoresis 1

Question 1

What is electrophoresis?

Answer 1

methods used for the separation and analysis of mixtures

- defined as the migration of a charged particle in an electric field

Question 2

How does separation occur in electrophoresis?

Answer 2

exploits the mass to charge ratio

an electric field applied across a medium in which the ions are placed in will cause them to migrate at different rates according to the ratio

at constant velocity the driving force on a particle is given by 
- product of the charge 
- applied field strength 
this is balanced by 
- frictional resistance of the medium

Question 3

What is ionic mobility?

Answer 3

ionic mobility/electrophoretic mobility
- given by the distance travelled in time under the influence of a potential gradient

electrophoretic mobility is directly proportional to distance travelled

Question 4

What are zwitterions?

Answer 4

molecules with both anionic (-) and cationic (+) groups

Question 5

What happens when you place zwitterions in basic and acidic media?

Answer 5

basic
- the carboxylic group loses a proton and the molecule gets an overall negative charge

acidic
- the amino group gains a proton and the molecule gets an overall positive charge

Question 6

What is ionic strength?

Answer 6

measure of the number of ions in solution
- made up of all the ions in solution

is affected by buffers

Question 7

How does ionic strength affect electrophoretic mobility?

Answer 7

mobility is inversely proportional the square root of ionic strength

buffers of low ionic strength have higher rates of migration than buffers of high ionic strength

Question 8

What is joule heating?

Answer 8

during electrophoretic separation, electrical energy is transformed into heat
- process is known as joule heating

Question 9

What are the side effects of joule heating? How can choice of buffer affect?

Answer 9

loss of water from the gel
decomposition of the analytes
damage to the apparatus

buffers affect ionic strength
increased ionic strength leads to increased conductivity which increases heat generated

Question 10

What gel is used in electrophoresis? What properties must it have?

Answer 10

polyacrylamide gel

must be
- chemically inert = are non-ionic
- have uniform matrix properties (pores)
= prevents separation based on the gel rather than the proteins

Question 11

What is the purpose of polyacrylamide gel?

Answer 11

gel prevents the convection observed in the liquid supports (the liquid the sample is in)
- water cannot be used as diffusion would occur

Question 12

How can polyacrylamide gel be manufactured?

Answer 12

is manufactured by the vinyl polymerisation of acrylamide monomers to form a long polyacrylamide chain
- the polymer is crosslink via the inclusion of a difunctional acrylamide

Question 13

What is the sieving effect

Answer 13

occurs when the passage of particles is hindered by the structure of the matrix (pores)

Question 14

How can pore size of polyacrylamide gel be altered/controlled?

Answer 14

by altering the concentration of the difunctional monomer
- difunctional acrylamide

increasing concentration of the difunctional monomer increases crosslink density which results in reduced pore size

Question 15

What are the factors affecting separation in PAGE?

Answer 15

pH - affects ionic/electrophoretic mobility
pore size of the gel - affects migration rate
electrode potential (E)
ionic strength

Question 16

How can you obtain separation based solely on charge?

Answer 16

use a large pore size

- removes sieving effects

Question 17

What is the function of tracking dye?

Answer 17

it makes the sample denser so it sits in the wells and does not float

migrates at a faster rate than the analytes and is visible
- allows the termination of electrophoresis to be seen

Question 18

What are the different detection methods?

Answer 18

uses stains to visualise bands
- stain is not bound so proteins and washes off leaving the corresponding bands

amino-black
coomassie blue
fast green