2. IEF (isoelectric focusing)
• In IEF proteins are separated according to their net charge
• The separation can be carried out in IPG (immobilized pH gradient) strip, tube gel or solution
• pH gradient is created with soluble ampholytes (polycarboxylic acid compounds) which
• When pH<pI protein becomes positively charged
and migrate towards cathode
• When pH=pI (isoelectric method) protein stops
moving
3. Creating pH gradient
• Using ampholytes
• Problems with ampholytes:
• Cathodic drift
• Reproducibility
• Using IPG strips
4. Chromatography in protein separation
• A separation technique that separates a mixture between fixed solid phase and a free moving
mobile phase is known as chromatography.
• Liquid chromatography is regularly used for protein separation.
Affinity Chromatography
5. Ion exchange chromatography
• Separates proteins on the basis of surface charge
distribution
• Protein is eluted from column by salt gradient
6. Reverse phase chromatography
• Reversible adsorption of protein
• Protein is eluted according to their hydrophobicity
• Matrix used: C4-C18 carbons
7. Size exclusion chromatography
• Separates proteins on the basis of
molecular weight
Multidimensional liquid
chromatography
• Liquid chromatography is used upstream or
downstream 2D-GE
• Combining the methods many problems can be
overcome
9. Protein sequencing techniques
Protein identification with antibodies:
• Antibodies identify special structural parts, epitopes
• Antibodies raised against a specific protein can be used
• Different techniques that use antibody: affinity chromatography, co-
immunoprecipitation, ELISA
• Limitation is unique antibody is required against each target protein
N-terminal sequencing:
• Chemical degradation of proteins: by boiling proteins in highly concentrated
hydrochloric acid for 24-72 hours
• Amino acids are labelled with reagents such as ninhydrin or fluorescamine
• Separated by HPLC
• Identified as they elute from the column, using standard amino acids as
reference
• N-terminal residue can be identified using dansyl chloride that reacts with
terminal amine group
10. Protein sequencing by Edman degradation
• Edman sequencing method was developed by Pehr Edman between 1960 and 1967
• N-terminal amino acid is labelled with phenyl isothiocyanate, followed by mild acid
hydrolysis
• This results in cleavage of the modified N-terminal (phenylhydrodantoin derivative) residue
• The terminal residue can be identified by HPLC
• Time consuming method: 24H for a 10 residue peptide, 3 days for a 30-40 residue peptide
• Problem with Edman degradation: time consuming, not functional if the N-terminal amino
acid is modified
