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• Solubility
• Molecular size, weight, density
• Affinity
• Charge
Solubility
Change in pH
Isoelectric precipitation
A procedure in which the pH of the protein mixture is adjusted to
the pI of the protein to be isolated to selectively minimize its
solubility.
Isoelectric precipitation
Change in ionic strength
Salting in
Solubility of a protein at low ionic strength generally
increases with the salt concentration.
Salting out
Decrease in solubility of proteins and other
substances in aqueous solution at high ionic
strength. It is a result of the competition
between added salt ions and other dissolved
solutes for molecular solvation.
Based on Molecular Size
Centrifugation
Process of subjecting a suspension of sample at greatly increased
gravitational field (centrifugal force) by rapidly rotating a receptacle
containing the sample which will lead to sedimentation of particles.
Application:
Differential Centrifugation
For separation of crude mixtures of cellular components
Dialysis
Itis the movement
of molecules by
diffusion from high
concentration to
low concentration.
Types of Gel:
1. Agarose
2. Polyacrylamide
SDS-PAGE
SDS: mask the intrinsic
charge of protein due
to large negative
charge it imparts on
it.
Separates protein in
the order of their
MWs.
Ion Exchange
Chromatography
Similarto affinity
chromatography
Interaction is based
on net charge
Column is packed
with resin that have
ligand (either positive
or negative in
charge)
Anion
Exchanger
Cation
Exchanger
Determination of 10 Structure of Protein
A. Qualitative and Quantitative Analysis of
Amino Acids
1. Hydrolyze peptide with 6N HCl at 110 C for 24 hours
2. Separate mixture by an amino acid analyzer
B. Determination of Amino Acid Sequence
1.Methods for identification of N-terminal amino acid
residue
a. Sanger’s Reagent (DNFB)
b. Dabsyl chloride and Dansyl chloride
c. Edman Degradation
d. Aminopeptidase