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: 2D-Electrophoresis

Practice: Use the results of the two-dimensional electrophoresis gel below to answer the following questions. 

A) Which protein or proteins have the highest pI value?

a. Protein a.                                    b. Proteins d & e.

b. Proteins b & c.


B) Which protein or proteins have the highest molecular weight?

a. Protein a.                                    c. Protein c.

b. Protein b.                                    d. Proteins d & e.


C) Which protein or proteins have identical molecular weights?

a. Proteins a & d.                           c. Proteins d & e.

b. Proteins b & c.                           d. None. Each has a unique weight.

Practice: Which of the following is true in 2D-electrophoresis?

Practice: An average protein will not be denatured by:

Practice: The first step in two-dimensional gel electrophoresis generates a series of protein bands by isoelectric focusing. In a second step, a strip of this gel is turned 90 degrees, placed on another gel containing SDS, and electric current is again applied. In this second step:

Practice: Sketch the result of 2D gel electrophoresis on the following four proteins (see chart) & label them clearly.