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

Practice: By adding SDS to a protein and performing gel electrophoresis, it is possible to:

Practice: True or false: Protein subunits linked via disulfide bonds appear as separate bands on an SDS-PAGE gel.

Concept #3: Visualizing Protein Purification on SDS-PAGE Gels

Practice: Which of the following statements are true regarding the treatment of proteins with SDS?

i) Only proteins with native net charges acquire an overall net negative charge.

ii) Proteins denature due to a disruption of the hydrophobic interactions stabilizing the core of their structures.

iii) All protein subunits can be separated via SDS-PAGE.

Practice: Suppose you purify a protein from liver cells and the SDS-PAGE results after different purification steps are shown. You then take the affinity purified sample and run it through a cation exchange column. The 2nd SDS-PAGE shows the results for the flow through and eluate from the cation exchanger.  Based on this data, what conclusions can you draw from the results in lanes #5, 7 & 8?


Lane #5:


Lane #7:


Lane #8: