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: Size-Exclsuion Chromatography

Practice: In a mixture of 5 proteins (listed below), which protein elutes 2 nd in size-exclusion chromatography?

Practice: Which of the following statements is false?

Practice: A new protein of unknown structure has been purified & gel filtration chromatography reveals that the native protein has a molecular weight of 240 kDa. Chromatography in the presence of 6 M guanidine hydrochloride (GuHCl), a chaotropic agent that has a similar effect on proteins as urea, yields a single absorbance peak corresponding to a protein of Mr 60 kDa. Chromatography in the presence both of 6 M guanidine hydrochloride and 10 mM β-mercaptoethanol (β-ME) yields peaks for proteins of Mr 34 kDa and 26 kDa. Using this data, which option best describes the structure of this protein? Hint: sketch a visual of the protein after each chemical treatment.

Practice: To answer the questions A, B & C below, use the provided chart with the properties of the four proteins.


A) What is the order of elution of the proteins from a size-exclusion chromatography column?

     a. A → B → C → D.

     b. D → B → A → C.

     c. B → D → A → C.

     d. C → A → D → B.


B) Which pH is best for separating the proteins using anion-exchange chromatography?

     a. pH = 6.                        b. pH = 7.                  c. pH = 8.


C) In what order would the proteins elute from the anion-exchange chromatography column?

     a. A → C → D → B.

     b. D → A → B → C.

     c. B → D → C → A.

     d. C → B → D → A.