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Allows:
Analysis of the biological properties
Understand its structure
Study interactions
Debye-Huckel Theory
Ammonium sulphate precipitation
Hemoglobin Myoglobin
Log of
Solubility
g mL-1
Fibrinogen Serum
albumin
1 2 3
Molarity of AmSO4
1M 2M 3M
AmSO4
The concept of a Column
Ion Exchange Chromatography (IEC)
Thus proteins are charge molecules and can have a different charge
at a given pH because they have different compositions of ionizable
amino acids
For any given amphoteric protein, there will be a pH at which
its overall charge is 0
poly-hisitidine
Chromatofocussing:
-Fractionates based on a pH gradient generated in an ion-
exchange column
HPLC:
-Similar to IEF and GFC, but uses pressure to increase
flow rates in columns with small particle size to increase
resolution of peaks
Methods for Assessing Protein Purity
SDS-PAGE Commonest method, rapid and sensitive
SDS
sodium dodecyl sulphate
PAGE
polyacrylamide gel electrophoresis
Migration of a molecule
in a electric field
Separates materials
based on size
Isoelectric focusing (IEF) can also be used, which separates
proteins by charge differences (induced by a pH gradient)
2D gel electrophoresis Combination of SDS-PAGE and IEF
Separates by charge in the first dimension and then by size in the
second dimension
Mass spectrometry
Protein Purification
Objectives
Purity
Stable
Cost
Time