Protein Tools:

Students will be able to:

Explain the techniques used to purify proteins,
The chemical principles by which the technique works
The different considerations to take into account when
using each

Know the techniques used to quantify and analyze

proteins.

Protein Purification
Requires a Strategy

Purified Proteins
Provide the Material for
Detailed Structural and Functional Analysis

Physical Characteristics
Distinguishing Proteins

Page 97

Column Chromatography
Mobile
Phase

Load
Buffer

(Stationary Phase)

Elution
Buffer

Isoelectric Points of
Several Common Proteins

Table 5-2

Ion-exchange
Chromatography

Ion Exchange Chromatography

Cation Exchanger:

Anion Exchanger:

Negatively charged
column binds positively
charged proteins

Positively charged
column binds negatively
charged proteins

Load Buffer pH < pI

Load Buffer pH > pI

Gel Filtration
Chromatography

Affinity Chromatography:
Most common purification strategy
Ligand:
Substrate adduct
or analog
High binding
affinity to protein
Non-reactive

Affinity Tags
protein

Uses known protein ligand pair

column

Target
Protein

ligand
column

OR:

Target
Protein

Encoded in DNA or otherwise covalently attached to

the protein
Elute with the ligand
Must be removed from protein, or check functionality

Examples of Affinity Tags

His-tag
(unique case, but most common)

GST-tag:
Column Ligand

Protein Favorite
(Affinity Tag) protein

Which of the following proteins will elute last in

gel filtration chromatography?
A.
B.
C.
D.

Proteinase Inhibitor III

Cytochrome c
RNA polymerase
Triose Phosphate Isomerase

3.4 kD
11.6 kD
98.8 kD
53.9 kD

If you want to perform an anion-exchange

purification of a protein which has a pI of 7.5,
what pH will you choose for this experiment?

A. pH 6.0
B. pH 7.0
C. pH 7.5

D. pH 8.0
E. pH 9.0

You are attempting to purify protein X by adding a biotin

tag to it. The protein Avidin binds very tightly to biotin.
How could you elute protein X off the affinity column?
I. Avidin in the elution buffer
II. Biotin in the elution buffer
III. Salt in the Elution buffer
A. I
B. II
C. I, III
D. II, III
E. I, II, III

Protein Analysis
Gives you information,
but are NOT purification techniques

Protein Assays:
Determines Protein amount
(1) General for total protein content

Protein binding Dyes

(2) Specific for the protein of interest

Antibody based assay

Absorbance spectroscopy

SDS-PAGE:
(PolyAcrylamide Gel Electrophoresis)
[CH3(CH2)10CH2OSO3]Na+
Sodium Dodecyl Sulfate

(SDS)

Separates proteins by size:

CANNOT be used for purfication,
CANNOT recover functional protein.

Figure 5-9

Measure Secondary Structure:

Indication of folded protein
Circular Dichroism (CD): Sample absorbs right- and leftcircularly polarized light to a different extent

Enzyme Activity Assay:

Purified protein is active
Analyze protein function
Experiment:
(1) Mix enzyme + substrate
(2) Record rate of product formation or reactant disappearance as a function of time

(the rate of reaction)

(3) Compare to previous data
New purification
[R]
Mutant protein
Different reaction conditions

[P]
Time

Why can you not use SDS-PAGE to purify proteins?

A. The SDS denatures the proteins
B. It separates protein by size
C. You cannot load a sufficient amount of
protein on a gel.
D. A and C
E. You can use SDS-PAGE to purify proteins