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Chapter Five Protein Purification and Characterization Techniques

This document provides an overview of protein purification and characterization techniques. It discusses the importance of purifying proteins and outlines general guidelines for protein purification, including defining objectives and properties of the target protein. It also discusses determining purity levels needed for different applications, various separation techniques based on physical and chemical properties, and analytical methods for tracking proteins during purification, including measuring activity, size-exclusion chromatography, electrophoresis, and isoelectric focusing. The overall goal of purification is to remove contaminating proteins while retaining the target protein.

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Reizelle Joy Billones
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164 views23 pages

Chapter Five Protein Purification and Characterization Techniques

This document provides an overview of protein purification and characterization techniques. It discusses the importance of purifying proteins and outlines general guidelines for protein purification, including defining objectives and properties of the target protein. It also discusses determining purity levels needed for different applications, various separation techniques based on physical and chemical properties, and analytical methods for tracking proteins during purification, including measuring activity, size-exclusion chromatography, electrophoresis, and isoelectric focusing. The overall goal of purification is to remove contaminating proteins while retaining the target protein.

Uploaded by

Reizelle Joy Billones
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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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

Why purify a protein?


Characterize function, activity, structure
Use in assays
Raise antibodies
many other reasons ...

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 AC

How pure should my protein be?

Application
Therapeutic use, in vivo
studies

Required Purity
Extremely high > 99%

Biochemical assays, X-ray


crystallography

High 95-99%

N-terminal sequencing,
antigen for antibody
production, NMR

Moderately high < 95%

Separation of proteins based on physical


and chemical properties
Solubility
Binding interactions
Surface-exposed hydrophobic residues
Charged surface residues
Isoelectric Point
Size and shape

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?

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 mol min-1
Specific activity
The specific activity is the activity of an enzyme per
milligram of total protein
expressed in mol min-1mg-1.
Specific activity is equal to the rate of reaction x
volume of reaction / mass of total protein.

How We Get Proteins Out of Cells

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
dont want to inhibit and enzyme activity when need to
assay during the purification

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

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

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

Column Chromatography

Ion Exchange
Interaction based on overall charge
(less specific than affinity)

Cation exchange

Anion exchange

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.

Size Exclusion/Gel-filtration (Contd)

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

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

Electrophoresis (Contd)
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)

SDS PAGE to track your purification

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

2D gel Size and Isoelectric point

Silver or commassie blue stain ---- Sypro Ruby - fluorescent

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

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