3. o Preparation for protein isolation
o Protein extraction and solubilization
o Protein concentration determination
o Concentrating protein solution
o Ammonium sulfate precipitation
o ultrafiltration, Freeze-drying (Lyophilization) , dialysis
o Column chromatography for purification
of protein
Steps of protein purification
17. Summary of affinity chromatography
Affinity chromatography is simple to do and can give
high purity in one step
Group-specific ligands are the easiest to use
Design the elution scheme
General elution conditions are often harsh
Specific eluents are kinder
18. Analytical tools
A rapid and reliable assay for the target
protein
Purity determination
e.g. SDS-PAGE
Total protein determination
e.g. colorimetric method
37. Primary sequence information and
amino acid composition from blots (Blotting
to PVDF)
1. Blotting is a technique for the electrophoretic transfer of DNA, RNA or
protein to a suitable membrane.
2. For protein sequencing and amino acid analysis, the proteins are
transferred to polyvinylidene difluoride (PVDF) membrane.
3. Bands visualized on the blot with Coomassie Brilliant Blue R-250 are
excised, cut into smaller pieces, washed extensively with deionized
water and used directly for N-terminal sequencing or amino acid
analysis