very simple view of protein purification which is a small component of the course here (chem 361). Mostly from Campbell 6th ed. with a small bit added on 2D gels.

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