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Affinity
Chromatography
Wenbo Dong
Yagmur Yagdiran
Technique
Charge
Size
Hydrophobicity
Affinity (AC)
Affinity chromatography
separates proteins on the basis
of a reversible interaction
between a protein (or group of
proteins) and a specific ligand
coupled to a chromatography
matrix.
Metal-Chelate Affinity
Chromatography
MetGlySerSerHisHisHisHisHisHisSer
SerGlyLeuValProArgGlySer....recomb
inant protein sequence
Theory Model
Exact model has not been established.
Langmuir Model
P + n Cu
==== P Cu + (n-1) Cu
Applications
Isolation and purification of denaturing
protein
Purification of enzyme
Purification of nucleotides
Analysis of Protein
Application of MCAC in other fields
Advantages
Two main advantages for using IMAC
Efficiently separating Histagged proteins in the
presence of denaturing concentrations of urea
and guanidineHCl
purification and the subsequent refolding can be
done in a single step
Disadvantag
es
the presence of metalions contaminats the purified
protein solution, because they may whether
destabilize or stabilize the protein