Working with Proteins
Introduction
• Our understanding of protein structure and function has been derived
from the study of many individual Proteins
• To study a protein in detail, the researcher must be able to separate it
from other proteins in pure form and must have the techniques to
determine its properties.
• Studying proteins and peptides sometimes requires purification from a
mixture
• Proteins contain different amino acid sequences.
• The sequence and arrangement of amino acids gives the protein a
chemical character
• Therefore, protein separation relies on:
– Charge
– Size
– Affinity for a ligand
– Solubility
– Hydrophobicity
– Thermal stability
Proteins Purification
Why do we need to purify a protein?
1. To determine its sequence
2. To determine its structure
3. To study its function
4. To analyze its physical properties
5. For industrial or therapeutic applications
, Sources of Proteins for Purification
• Animal tissues
• Plant tissues
• Recombinant DNA technology
Steps involved in purification of a typical soluble
protein
• Cell lysis: opening cells to release their proteins into a solution called
crude extract
• Differential Centrifugation- cell free extract
– Used to remove membranes, nuclei, large organelles etc.
• Fractionation
– Based on differences in protein size, charge, binding affinity etc.
o Column Chromatography
• Detection:
• Electrophoresis
o Column Chromatography general steps
• Protein binding to a matrix
• Washing
• Elution/release
Protein purification: Column chromatography
The most powerful method for fractionation:
Solid Phase (Matrix):
porous material (glass or plastic) supported inside a column
Introduction
• Our understanding of protein structure and function has been derived
from the study of many individual Proteins
• To study a protein in detail, the researcher must be able to separate it
from other proteins in pure form and must have the techniques to
determine its properties.
• Studying proteins and peptides sometimes requires purification from a
mixture
• Proteins contain different amino acid sequences.
• The sequence and arrangement of amino acids gives the protein a
chemical character
• Therefore, protein separation relies on:
– Charge
– Size
– Affinity for a ligand
– Solubility
– Hydrophobicity
– Thermal stability
Proteins Purification
Why do we need to purify a protein?
1. To determine its sequence
2. To determine its structure
3. To study its function
4. To analyze its physical properties
5. For industrial or therapeutic applications
, Sources of Proteins for Purification
• Animal tissues
• Plant tissues
• Recombinant DNA technology
Steps involved in purification of a typical soluble
protein
• Cell lysis: opening cells to release their proteins into a solution called
crude extract
• Differential Centrifugation- cell free extract
– Used to remove membranes, nuclei, large organelles etc.
• Fractionation
– Based on differences in protein size, charge, binding affinity etc.
o Column Chromatography
• Detection:
• Electrophoresis
o Column Chromatography general steps
• Protein binding to a matrix
• Washing
• Elution/release
Protein purification: Column chromatography
The most powerful method for fractionation:
Solid Phase (Matrix):
porous material (glass or plastic) supported inside a column
