Polyacrylamide gel electrophoresis (PAGE) of proteins

Question 1

What is electrophoresis?

Answer 1

movement of charged molecules in response to an electric field resulting into their separation

Question 2

In free solution, what is mobility dependent on?

Answer 2

Mainly on net charge

Question 3

When gel is present, what is mobility dependent on?

Answer 3

Size and shape of protein

Question 4

What is the relationship between charge and mobility?

Answer 4

Higher the charge, higher the mobility

Question 5

For electrophoresis, where do negative charges attracted towards? Positive charges?

Answer 5

Negative charge moves towards the anode (+)

Positive charge moves toward the cathode (-)

Question 6

What is polyacrylamide gel?

Answer 6

Cross linked polymers of acrylamide and bis-acrylamide

Question 7

What are the size of the pores in polyacrylamide gel dependent on?

Answer 7

total concentration (%T, w/v) of acrylamide and bis-acrylamide and % Cross linkers (w/w)

Usually the separating gel % varies between 4-20%

Question 8

What is electrophoretic mobility dependent on?

Answer 8

Charge, molecular weight, charge to mass ratio, gel concentration and viscosity

Higher electric field, higher mobility

Question 9

What is the Ferguson plot?

Answer 9

a relationship between the concentration of gel and the electrophoretic mobility of a protein

Same charge but different mass

All proteins extrapolate the same mobility at 0% gel

Question 10

What are the two types of PAGE?

Answer 10

Native-PAGE (non-denaturing)

SDS-PAGE (denaturing)

Question 11

What is native-PAGE?

Answer 11

Separation based on native charge, size, and shape

Question 12

What is SDS-PAGE?

Answer 12

Separation is purely based on size

SDS will make proteins negative so size is the only variability (native charged masked)

SDS denatures the protein

Question 13

How does SDS denature the protein?

Answer 13

Dissociates subunits

Unfolds polypeptide chains (linear)

Binds to denatured protein and imparts negative charge to the polypeptide chain

Question 14

If a native-PAGE shows a band high in the gel and then in SDS the band goes down the gel, what does this mean?

Answer 14

Shows that this is made up of 2 subunits SO

Protein is most likely a homodimer with non-covalent bonds

Question 15

What do reducing agents do?

Answer 15

break down the disulfide bonds

B-mercaptoethanol (2-ME) and Dithiothreitol (DTT)

Question 16

What is the relationship between mobility and gel concentration?

Answer 16

Mobility inversely proportional to gel concentration (Ferguson chart)

Bigger size, lower mobility

Question 17

How is the separation pattern of proteins different under non-reduced and reduced conditions of SDS-PAGE?

Answer 17

In non-reduced conditions, disulfide bond does not break, stay as a dimer, and the molecular weight stays the same as native

In reduced conditions, disulfide bond gets cleaved and now are monomers which makes two subunits of half the native weight. It also goes down the gel.

Question 18

How to get molecular weight of protein?

Answer 18

Using Rf

Rf = distance migrated by the protein / distance migrated by the dye front

Values range from 0-1