Sections
Protein Purification
Protein Extraction
Differential Centrifugation
Salting Out
Dialysis
Column Chromatography
Ion-Exchange Chromatography
Anion-Exchange Chromatography
Size Exclusion Chromatography
Affinity Chromatography
Specific Activity
HPLC
Spectrophotometry
Native Gel Electrophoresis
SDS-PAGE
SDS-PAGE Strategies
Isoelectric Focusing
2D-Electrophoresis
Diagonal Electrophoresis
Mass Spectrometry
Mass Spectrum
Tandem Mass Spectrometry
Peptide Mass Fingerprinting
Overview of Direct Protein Sequencing
Amino Acid Hydrolysis
FDNB
Chemical Cleavage of Bonds
Peptidases
Edman Degradation
Edman Degradation Sequenator and Sequencing Data Analysis
Edman Degradation Reaction Efficiency
Ordering Cleaved Fragments
Strategy for Ordering Cleaved Fragments
Indirect Protein Sequencing Via Geneomic Analyses

Concept #1: Ion-Exchange Chromatography

Concept #2: Cation-Exchange Chromatography

Practice: What is the order of elution of the following proteins from a cation-exchange chromatography column? 

Net charges of Proteins: Protein A = +1              Protein B = -2                Protein C = -5                Protein D = +3.

Practice: In a cation-exchange column at neutral pH, which peptide would elute last?

Practice: Mixtures of amino acids can be analyzed by first separating the mixture into its components through ion exchange chromatography. Certain amino acids placed on a cation-exchange resin containing sulfonate groups (—SO3-) flow down the column slowly because of two factors that influence their movement: (1) ionic attraction between the sulfonate residues on the column and positively charged functional groups on the amino acids, and (2) hydrophobic interactions between amino acid R-groups and the strongly hydrophobic backbone of the polystyrene resin. For each pair of amino acids listed below, circle the amino acid that is eluted first from the cation-exchange column by a buffer at pH 7.

Practice: Give the order of elution of the following peptides when using cation-exchange chromatography at pH 7.2.

Peptide #1: A-D-G-H-E.           Peptide #2: K-L-M-R-A.           Peptide #3: M-D-L-I-V.             Peptide #4: I-L-R-P-M.


Order of Elution: _______________, _______________, _______________, _______________

                                 (1   st to elute)                                                                             (Last to elute)