Chromatography

Question 1

Chromatography

Answer 1

• Separation based on affinity for mobile phase vs. stationary phase

Question 2

Size Exclusion (Gel Filtration)

Answer 2

• Separation based on size
  
  • Large proteins elute first

Question 3

Ion Exchange Chromatography

Answer 3

• Separation based on charge
  
  • Anion Exchange
    
    • Anions bind to + charges
  • Cation Exchange
    
    • Cations bind - charges

Question 4

Hyrdrophobic Interaction

Answer 4

• Stationary phase contains hydrophobic groups
  
  • Separation based on hydrophobicity of the protein

Question 5

Affinity Chromatography

Answer 5

• Stationary phase contains specific ligand
  
  • Elute with excess ligand

Question 6

Native Electrophoresis

Answer 6

Separation based on natie charge of proteins

Question 7

SDS-PAGE

Answer 7

• Separation based on size
• SDS- Sodium Dodecyl Sulfate
  
  • Interacts with proteins at approximately equal-charge-to-mass rations
  • Disrupts non-covalent interaction of 4° structure
• Smaller proteins migrate faster

Question 8

Isoelectric Focusing (IEF)

Answer 8

• Buffers used to generate a pH gradient
  
  • Separation based pI

Question 9

Two-Dimensional Gel Electrophoresis

Answer 9

• Isoelectric Focusing in first dimension/SDS-PAGE in second dimension

Question 10

Western Blot

Answer 10

• Separted proteins that are incubated with and antibody initiating an antibody-antigen reaction

Question 11

Edman Sequencing

Answer 11

1. Chemical procedure removes one amino acyl residue from N-terminus
2. Amino acyl residue can be identified
3. Residue sequence can then be determined
4. Database allows for protein identification

Question 12

Mass Specrometry

Answer 12

• Molecules are separated by mass w/ very high sensitivity
• For proteins
  
  • Subject to trypsin
  • Allows for identification of cleaved polypeptides
    
    • Detects covalent modications