Protein Isolation & Analysis Techniques

Question 1

3 kinds of electrophoresis

Answer 1

Native PAGE, SDS PAGE, Isoelectric focusing

Question 2

Native PAGE

Answer 2

• maintains the protein’s shape
• proteins separated in electric field on basis of net negative charge/size
• smaller and more negatively charged proteins migrate faster through gel
• results difficult to compare because mass-to-charge ratio differs for each protein

Question 3

SDS-PAGE

Answer 3

• used to determine degree of purity, molecular mass, and number of polypeptide subunits in protein
• sample is treated with reducing agent to break disulfide bonds and then SDS (an anionic detergent) denatures and covers proteins with an overall negative charge
• proteins all have identical mass-to-charge ratio after treatment with SDS, thus they are separated on the basis of their mass
• smallest proteins move farthest

Question 4

Isoelectric focusing

Answer 4

• proteins separated by their pI
• pH gradient applied to gel
• each protein migrates through gel until it reaches the point where it has no net charge (its pI)

Question 5

Chromatography

Answer 5

separates protein mixtures on the basis of their affinity for a stationary or mobile phase

Question 6

column chromatography

Answer 6

uses beads of a polar compound as stationary phase with nonpolar solvent as mobile phase

Question 7

ion-exchange chromatography

Answer 7

• proteins separated on basis of their net charge
• uses charged column and variably saline eluent
• anion exchange and cation exchange

Question 8

anion exchange chromatography

Answer 8

column contains positive beads to which negatively charged proteins bind

Question 9

cation exchange chromatography

Answer 9

column contains negatively charged beads to which positively charged proteins bind

Question 10

size exclusion chromatography

Answer 10

• relies on porous beads

- larger molecules elute first because they are not trapped in small pores

Question 11

affinity chromatography

Answer 11

uses a bound receptor or ligand and an eluent with a free ligand or a receptor for the protein of interest

Question 12

Protein structure is primarily determined through:

Answer 12

X-ray crystallography… although NMR can also be used