Lecture 7: Protein Purification and Identification

Question 1

Importance of protein purification

Answer 1

• Other molecules may interfere/modify the protein, creating a heterogenous population
• Purifying a protein is first step in understanding structure or function

Question 2

Cell lysis

Answer 2

• Methods: Grinding, sonication, vortexing with glass beads, detergents
• Lysing cells may release proteases, enzymes that cleave POI. Protease inhibitors can prevent degradation of POI

Question 3

Centrifugation

Answer 3

• Centrifugation can be used to seperate supernatant of soluble materials from pellet of other insoluble precipitants
• Used to isolate particular organelle, cellular compartment

Question 4

Beer-lambert law

Answer 4

A = Ɛcl

Ɛ = molar extinction coefficient
c = concentration
l = path length

Question 5

Chromatography

Answer 5

• Differential partitioning of a molecule between a mobile and stationary phase
• Proteins can be purified based off differences in chemical properties
• Size/shape: Size Exclusion/Gel filtration
• Charge: Ion exchange
• Binding interactions: Affinity
• Hydrophobicity: HPLC

Question 6

Size Exclusion Chromatography

Answer 6

• Proteins separated based on size
• Columns contain resin of beads(smaller proteins get stuck, while larger move quickly)
• Calibration with proteins of known weight is required

Question 7

Ion Exchange Chromatography

Answer 7

• Separates amino acids based on net charge
• Cation exchange resins attract and bind positively charged polypeptides
• Anion exchange resins attract and bind negatively charge polypeptides
• Proteins can be eluted by changing pH

Question 8

Affinity Chromatography

Answer 8

• Proteins are attracted to columns based on affinity for specific molecules or chemical groups
• Different resin contain covalently bound molecules or ligands that are complementary to certain proteins
• Bound protein is released from passing a solution containing free molecules to compete for binding

Question 9

Dialysis for protein purification

Answer 9

• Dialysis can be used to remove small molecules that were used to elute proteins from a column

Question 10

High Pressure Liquid Chromatography(HPLC)

Answer 10

• Uses fine beads and high-pressure pump to move a sample through column achieving higher resolution of peaks
• Also known as Reverse Phase HPLC when separating based on hydrophobicity
• Hydrophobic compounds interact stronger with column and have longer retention time

Question 11

SDS-Page

Answer 11

• SDS is a detergent and denatures protein
  
  • Gives a polypeptide a unform - charge
• BME can be used to reduce disulfide bonds
• Polypeptide chains will have same mass-to-charge ratio and migrate towards anode
• Size can be deduced by comparing to MW markers