Electrophoresis

Question 1

 A separation process by

Answer 1

applying an electric field

Question 2

 Under the influence of an electric field

Answer 2

dissolved ions present in a solution will migrate at varied rates and directions in a column or a
surface

Question 3

Ions of opposite charge will migrate in

Answer 3

different directions and become separated on that basis

Question 4

Ions of like charge, while migrating in the same direction,

Answer 4

become separated due to different migration rate

Question 5

The rates of migration is depending on:

Answer 5

(a) The strength of the electric field
(b) The net charge on the molecule
(c) The size of the molecule
(d) The viscosity of the medium through which the ions
travel

Question 6

Gel electrophoresis

Answer 6

Separations are carried out on a thin flat layer (slab) made of a porous semi-solid insoluble material such as agarose or acrylamide

Question 7

Gel electrophoresis example

Answer 7

DNA gel electrophoresis, SDSPAGE gel electrophoresis,

Question 8

Capillary electrophoresis (CE)

Answer 8

Separation happened in a buffer filled fused silica capillary that is typically 10 to 100 um in internal diameter and 40 to 100 cm long, extends between two buffer reservoirs that also hold platinum electrodes

Question 9

SDS is a detergent that

Answer 9

dissociates and unfolds oligomeric proteins
into its subunits

Question 9

SDS-PAGE

Answer 9

Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis

Question 10

The SDS binds to the polypeptides to form

Answer 10

complexes with fairly constant charge to mass ratios

Question 11

SDS is an anionic detergent, meaning that when dissolved

Answer 11

its molecules have a net negative charge within a wide pH range

Question 12

The negative charges on SDS destroy

Answer 12

most of the complex structure of proteins, and are strongly attracted toward an anode (positively-charged electrode) in an electric field

Question 13

The electrophoretic migration rate through a gel is therefore determined only by

Answer 13

y the size of the complexes

Question 14

In the native mode,

Answer 14

the mobility is affected by molecular weight, shape and charge

Question 15

In denaturing mode

Answer 15

 Sample is heated and treated in SDS solution, which denatures and bind to protein, to form a complex that makes the protein to have large negative charge
 SDS are attached to the protein in a constant ratio. Protein now have identical charge density
 The proteins are now ready to be separated based on the difference in shape
 Under these conditions, all the protein particles would migrate at the same rate,
through a matrix of various hole sizes, as exist in the gel
 The larger protein will interact with the gel structure, thus travel slower in the gel

Question 16

Polyacrylamide gels are formed by the reaction of

Answer 16

acrylamide and bis-acrylamide
(N,N’-methylenebisacrylamide) that results in highly cross-linked gel matrix

Question 17

The gel acts as a

Answer 17

sieve through which the proteins move in response to the electric field

Question 18

 Proteins contain an overall positive or negative charge; this enables

Answer 18

s the movement of a protein molecule towards the isoelectric point at which the molecule has no net charge

Question 19

By denaturing the proteins and giving them a uniform negative charge,

Answer 19

it is possible to separate them based on the size as they migrate towards the positive
electrode

Question 20

Smaller proteins migrate more quickly and

Answer 20

thus move further through the gel

Question 21

large molecules migrate only

Answer 21

a short distance

Question 22

In order to separate proteins on a gel solely according to size

Answer 22

all of the molecules must have the same charge-to-mass ratio

Question 23

After separation by electrophoresis,
protein on the gel cannot be seen

Answer 23

and only if staining is performed, the protein is coloured

Question 24

Molecular weights are determined simultaneously running

Answer 24

marker proteins of known molecular weight

Question 25

Estimation of molecular mass by

Answer 25

comparing the migration distance to that of a protein with a known mass

Question 26

Preparing a standard curve

Answer 26

To determine the molecular weight of the
proteins, one must run standard proteins of
known size on the same gel along with the
samples