Lecture 8: Protein Purification and Identification

Question 1

Identifying polypeptides

Answer 1

• SDS Page and Western Blotting can be used to confirm presence of larger polypeptides using antigen specific molecules
• Mass spectrometry can also identify polypeptides

Question 2

Western Blotting

Answer 2

• Proteins are separated by SDS Page and transferred to a solid-support membrane
• Membrane is exposed to a protein solution to bind random proteins to any unused regions of the membrane
• Primary antibody, specifically for POI is added to recognize
• Secondary antibodies are specific for Fc domain of primary antibody and are attached to fluorescently labeled tag
• Shows where POI is present in the sample and its approximate size based on where it migrated in gel

Question 3

Mass Spectrometry

Answer 3

• Peptides are bombarded by laser or high energy electron beam to create ionized fragments
• Products are attracted to a charged plate detector and analyzed in a mass analyzer
• Time of flight depends on charge and mass of molecule

Question 4

Protein Sequencing

Answer 4

• Sequence could be obtained using DNA sequencing
• Edman degradation is a chemical reaction based method that removes amino acids starting from N-terminus