Part 1 - Video Lecture Notes
Tuesday, February 5, 2019 4:42 PM
Column chromatography
• Fraction: a set of volume collected after every purification step.
○ Proteins that interact more with the stationary phase will be present in the later fractions.
○ Proteins that interact less with the stationary phase will be present in the earlier fractions.
• @ different pHs, proteins will have different charges.
○ pH > pI: proteins are (-) charged. Low [H] -> proteins will deprotonate into environment.
○ pH < pI: proteins are (+) charged. High [H] -> proteins will be protonated by environment.
• To determine which proteins are in which fractions, you would measure the activity of each fraction.
Protein that will elute in fractions…
Column Early Middle Late
Gel filtration Large Intermediate Small
Cation exchanger Anions Neutral ions Cations
Anion exchanger Cations Neutral ions Anions
Affinity Column Low affinity Not affected by ligands High affinity
Tuesday, February 5, 2019 4:42 PM
Column chromatography
• Fraction: a set of volume collected after every purification step.
○ Proteins that interact more with the stationary phase will be present in the later fractions.
○ Proteins that interact less with the stationary phase will be present in the earlier fractions.
• @ different pHs, proteins will have different charges.
○ pH > pI: proteins are (-) charged. Low [H] -> proteins will deprotonate into environment.
○ pH < pI: proteins are (+) charged. High [H] -> proteins will be protonated by environment.
• To determine which proteins are in which fractions, you would measure the activity of each fraction.
Protein that will elute in fractions…
Column Early Middle Late
Gel filtration Large Intermediate Small
Cation exchanger Anions Neutral ions Cations
Anion exchanger Cations Neutral ions Anions
Affinity Column Low affinity Not affected by ligands High affinity
