Lecture 6

Question 1

What happens in electrophoresis?

Answer 1

charged molecules are separated in an electric field

Question 2

The rate of molecule migration in electrophoresis depends on what five factors?

Answer 2

electrical field strength; net charge of particle; viscosity of electrophoretic medium; shape of molecule; interaction of molecule of interest with other molecules in the electrophoresis system

Question 3

Which electrophoretic medium is used to separate amino acids?

Answer 3

paper (cellulose polymer)

Question 4

Which electrophoretic medium is used to separate DNA/RNA?

Answer 4

agarose gel (carbohydrate polymer)

Question 5

Which electrophoretic medium is used to separate proteins?

Answer 5

polyacrylamide

Question 6

SDS is what type of detergent?

Answer 6

ionic detergent

Question 7

What stacker are we using?

Answer 7

5% stacker

Question 8

What resolver are we using?

Answer 8

12% resolver

Question 9

What is the acrylamide:bisacrylamide ratio?

Answer 9

29:1

Question 10

What converts the acrylamide monomer into a polymer? (2)

Answer 10

APS; TEMED

Question 11

Describe the SDS molecule structure (2)

Answer 11

negatively charged head; hydrophobic tail

Question 12

How many SDS molecules are needed per amino acid?

Answer 12

1 SDS molecule per 2 amino acids

Question 13

Give two examples of reducing agents

Answer 13

DTT or beta-mercaptoethanol

Question 14

What happens as you decrease resolver percentage?

Answer 14

Separation between masses increases, the smaller masses may not even show on the gel

Question 15

How do we plot the SDS PAGE standard curve?

Answer 15

logMW vs. distance travelled; curve is bi-phasic

Question 16

The band that travelled the least distance has what MW?

Answer 16

has the highest MW

Question 17

What is the purpose of the SDS in the system?

Answer 17

it denatures proteins and coats them to give an equal mass-to-charge ratio (essentially, removes the variability of charge in different proteins)

Question 18

What is the purpose of the glycerol in the sample-loading buffer?

Answer 18

Glycerol adds density to the sample, causing it to sink to the bottom of the well.

Question 19

What is the purpose of the bis-acrylamide?

Answer 19

If only acrylamide is present, then you create a linear polymer. The bis-acrylamide causes crosslinking of chains so it forms a “pore.”

Question 20

What happens if there’s no glycerol in the system?

Answer 20

less dense proteins will be higher in the well and denser proteins will be closer to the bottom of the well. Therefore, not all of the proteins will begin running through the gel at the same time.

Question 21

What happens if there’s no bis-acrylamide in the system?

Answer 21

The gel won’t have pores and nothing will control the migration of proteins – some may run off the gel

Question 22

Where does the y-axis of an SDS standard curve start?

Answer 22

10 (not 0, because log(0) is undefined)

Question 23

If you have a low MW you want to resolve (e.g. between 25 kDa and 12.5 kDa)

Answer 23

use a higher percentage resolver

Question 24

If you have a high MW you want to resolve (e.g. between 125 kDa and 120 kDa),

Answer 24

use a lower percentage resolver

Question 25

What is the purpose of bromophenol blue?

Answer 25

dye front

Question 26

What happens if there is no SDS in the system?

Answer 26

proteins will have different charge to mass ratios; positively charged particles will travel in reverse to negative electrode; shape of protein may also have impact bc it’s not denatured

Question 27

Design an experiment to determine if a protein is monomeric or oligomeric. If oligomeric, are they held together by disulfide bonds?

Answer 27

Run two samples of the protein on the same SDS-PAGE - purified protein with known MW in one lane, protein without reducing agent in the other lane