This document provides an overview of protein purification and characterization techniques, outlining the importance of defining objectives and properties of target proteins. It details various methods such as salting out, column chromatography, and electrophoresis, emphasizing the delicate nature of proteins and the need to track their activity during purification. Key metrics discussed include activity, specific activity, and the required purity for different applications.

1. Mary K. Campbell
Shawn O. Farrell
http://academic.cengage.com/chemistry/campbell
Chapter Five
Protein Purification and
Characterization Techniques
Paul D. Adams • University of Arkansas

2. Why purify a protein?
• Characterize function, activity, structure
• Use in assays
• Raise antibodies
• many other reasons ...

3. Guidelines for protein purification
• Define objectives
• Define properties of target protein and critical
contaminants
• Minimize the number of steps
• Use a different technique at each step
• Develop analytical assays
Adapted from: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition

4. How pure should my protein be?
Application Required Purity
Therapeutic use, in vivo
Extremely high > 99%
studies
Biochemical assays, X-ray
High 95-99%
crystallography
N-terminal sequencing,
antigen for antibody Moderately high < 95%
production, NMR

5. Separation of proteins based on physical
and chemical properties
• Solubility
• Binding interactions
• Surface-exposed hydrophobic residues
• Charged surface residues
• Isoelectric Point
• Size and shape

6. The overall goal
• To remove as much of the “other” protein as possible
and keep as much of your target protein as possible
• This is a great challenge since at each step you
sacrifice some of your target protein.
• Activity = total target protein activity in your sample
• Specific activity = how much target enzyme activity
you have with respect to total protein content present
• Which number should go up and which down?

7. Activity versus Specific Activity
Enzyme activity
• Enzyme activity = moles of substrate converted per
unit time = rate × reaction volume. Enzyme activity is
a measure of the quantity of active enzyme present
• 1 enzyme unit (U) = 1 μmol min-1
Specific activity
• The specific activity is the activity of an enzyme per
milligram of total protein
• expressed in μmol min-1mg-1.
• Specific activity is equal to the rate of reaction x
volume of reaction / mass of total protein.

8. How We Get Proteins Out of Cells

9. Proteins/enzymes are delicate
• Remember Proteins are delicate and subject to denaturation.
• Often tracking a protein based on its activity or function
therefore it needs proper conformation
• Cells are full of hydrolytic enzymes when you fracture or lyse a
cell proteins and enzymes are mixed and degradation occurs
immediately
• Keep things cold (on ice)
• Add protease inhibitors
• Many considerations to be made when using and selecting
protease inhibitors – remember the six classes of enzymes
– don’t want to inhibit and enzyme activity when need to
assay during the purification

10. How will you track your protein?
• Purification is often a multi-step process
• You need to track or “assay for your protein” after each
step
• If it is an enzyme you can test for its activity
• If you have an antibody you can use Western blot or
ELISA
• You can test for its size (not as specific)
• You could use mass spectrometry to identify it
• You could use N-terminal sequencing to ID the traget
protein

11. Salting Out
• After Proteins solubilized, they can be purified based
on solubility (usually dependent on overall charge,
ionic strength, polarity
• Ammonium sulfate (NH4SO4) commonly used to “salt
out”
• Takes away water by interacting with it, makes protein
less soluble because hydrophobic interactions among
proteins increases
• Different aliquots taken as function of salt
concentration to get closer to desired protein sample
of interest (30, 40, 50, 75% increments)
• One fraction has protein of interest

12. Column Chromatography
• Basis of Chromatography
• Different compounds distribute themselves to a varying
extent between different phases
• Interact/distribute themselves
• In different phases
• 2 phases:
• Stationary: samples interacts with this phase
• Mobile: Flows over the stationary phase and carries
along with it the sample to be separated

13. Column Chromatography

14. Ion Exchange
• Interaction based on overall charge
(less specific than affinity)
• Cation exchange
• Anion exchange

15. Size-Exclusion/Gel-Filtration
• Separates molecules based on size.
• Stationary phase composed of cross-linked gel
particles.
• Extent of cross-linking can be controlled to determine
pore size
• Smaller molecules enter the pores and are delayed in
elution time. Larger molecules do not enter and elute
from column before smaller ones.

16. Size Exclusion/Gel-filtration (Cont’d)

17. Affinity Chromatography
• Uses specific binding properties of molecules/proteins
• Stationary phase has a polymer that can be covalently
linked to a compound called a ligand that specifically
binds to protein

18. Electrophoresis
• Electrophoresis- charged particles migrate in electric
field toward opposite charge
• Proteins have different mobility:
• Charge
• Size
• Shape
• Agarose used as matrix for nucleic acids
• Polyacrylamide used mostly for proteins

19. Electrophoresis (Cont’d)
• Polyacrylamide has more resistance towards larger
molecules than smaller
• Protein is treated with detergent (SDS) sodium
dodecyl sulfate
• Smaller proteins move through faster (charge and
shape usually similar)

20. SDS PAGE – to track your purification

21. Isoelectric Focusing
• Isolectric focusing- based on differing isoelectric pts.
(pI) of proteins
• Gel is prepared with pH gradient that parallels electric-
field. What does this do?
• Charge on the protein changes as it migrates.
• When it gets to pI, has no charge and stops

22. 2D gel – Size and Isoelectric point
Silver or commassie blue stain ---- Sypro Ruby - fluorescent

23. Differential Centrifugation
• Sample is spun, after
lysis, to separate
unbroken cells, nuclei,
other organelles and
particles not soluble in
buffer used
• Different speeds of
spin allow for particle
separation