BIOL213 Lecture Notes - Lecture 3: Aromatic Amino Acids, Affinity Chromatography, Column Chromatography
Lecture 3 – Protein separation techniques
Separation strategies and techniques
Cells, tissues and body fluids contain thousands of proteins. Proteins extracted from
human epidermal cells are a crude extract.
A mixture of proteins can be separated, and this separation relies on differences in
physical and chemical properties:
• Solubility
• size
• charge
• Affinity for a ligand
• hydrophobicity
• Thermal stability
Chromatography is a commonly used first step for separating protein mixtures.
Steps for purifying protein from a crude extract:
1. “Salting out” and dialysis: ammonium sulphate can selectively precipitate some
proteins based on solubility. The precipitates are separated by low speed
centrifugation.
The protein solution is then dialysised to remove small molecular weight
components, such as salt ions.
2. Column chromatography: protein samples are separated based on how they move
through a matrix
• Spectroscopic detection of aromatic amino acids: the aromatic acids absorb light in
the UV region. Proteins typically have UV absorbance maxima around 275-
280nm.
Tryptophan and tyrosine are the strongest chromophores.
The concentration of the UV-visible light can be determined using Beer-Lambert
law.
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Cells, tissues and body fluids contain thousands of proteins. Proteins extracted from human epidermal cells are a crude extract. A mixture of proteins can be separated, and this separation relies on differences in physical and chemical properties: size: solubility, charge, affinity for a ligand, hydrophobicity, thermal stability. Chromatography is a commonly used first step for separating protein mixtures. Steps for purifying protein from a crude extract: salting out and dialysis: ammonium sulphate can selectively precipitate some proteins based on solubility. The precipitates are separated by low speed centrifugation. Proteins typically have uv absorbance maxima around 275- The concentration of the uv-visible light can be determined using beer-lambert law. Lecture 3 protein separation techniques: size-exclusion chromatography (gel filtration): the matrix is a synthetic polymer resin with pores of certain sizes. Small proteins can permeate through the pores however large proteins are excluded.
