Nonenzymatic protein function and analysis

Question 1

Electrophoresis separates by ____ and works by ____

Answer 1

Separates by charge and size

An electric field is generated, proteins move towards opposite charge (ex: + goes to - side)
-Small proteins move faster than large bc they can fit through porous gel better
-+ side on left, - side on right

Question 2

What is Native PAGE used for?

Answer 2

Analyzes in native state

Useful for comparing size/charge of proteins in analyzed in SDS PAGE first
-cannot be used if the protein is denatured
-can be difficult since so many proteins share similar sizes/charges/mass

Question 3

SDS-PAGE separates by ____ and works by ____

Answer 3

Separates by mass alone

Creates a net negative charge on all proteins so that they’re able to migrate by mass alone
-smaller proteins go farther than large ones

Question 4

Iso-electric focusing separates by ____ and works by ____

Answer 4

Separates by pI

A pH gradient is created vix electric current, then proteins travel until they reach their pI

Question 5

All chromatography’s separate by ____

Answer 5

Chemical and physical properties

Question 6

Column chromatography separates by _____ and works by ____

Answer 6

Size and polarity

Polarities that are most similar to the stationary phase elute first, since proteins that are least similar will be attracted to the SP and stick around longer
-Large compounds also elute first bc they’re too large to be trapped by the SP

Question 7

Ion-exchange chromatography separates by ____ and works by ____

Answer 7

Separates by charge

The charged SP will attract opposite charges, so charges LIKE the SP will elute first (since they have no desire to interact with the SP

Question 8

Size-exclusion chromatography separates by ____ and works by ____

Answer 8

Separates by size

The SP is porous, so larger proteins elute first since they will not get caught in the SP pockets

Question 9

Affinity chromatography separates by ____ and works by ____

Answer 9

Separates by chemical properties

SP is chosen so that it has a high affinity for specific protein. The protein will stick to the SP, all others will elute out of the column, then the target protein can be washed from the SP and collected