Protein Purification

and Characterization

Protein Purification and

Characterization

Why Study proteins?

IMPORTANT FACTORS IN PROTEIN PURIFICATION

Starting materials
tissues, cells or clones expressed in E. Coli or animal
cells
Decisions quantity of protein, protein modification availability of
samples, is it cloned yet, expense

Stabilization of protein is key - proteins are not

meant to be purified, so you need to keep
them alive and happy (active / native)

pH - both activity and structure are pH dependent

Temperature - most stabile at low temperature reduces energy in the system for unfolding and
reduces the protease kinetics. Few proteins are
unstable at low temps - ppdk (Dr. Chastain's enzyme)
and the ATPase in mitochondria
Protease inhibitors - several classes of proteins
catalyze the hydrolysis of peptide bonds (called
proteases). Usually need to add several "suicide"
inhibitors and reduce free metals which are used by
the proteases
Reducing agents - beta - mercaptoethanol and
dithiolthreitol both act as reducing agents. Prevent
the oxidation of amino acids
Detergents - Membrane bound proteins often need
added detergents (soaps) to mimic the ampipathic
nature of the membrane you so cruelly ripped it from
- need to be above the concentration at which
micelles are formed - the critical micellular
concentration (CMC)

Homogenization - breaking the cell apart

Mechanical Shearing - Warring blender, glass
or plastic pestle like homogenizer
Freeze Thaw - cycles under hypnotic
conditions, ice crystals disrupts membranes

Enzymatic degradation of the cell

membrane - mostly for
bacterial preparation
Sonication - high energy sounds
to disrupt membrane
Detergent disruption of memb

METHODS OF PURIFICATION
Centrifugation - Separation based on density, mass,
shape and the density of the solution
Sedimentation of particles measured by Svedburg
units
Force applied in gravitational (g) forces
The centrifugal force depends on speed and time and
radius of rotor
Differential centrifugation
One of the most used methods in biochemistry
Uses increasing g forces to yield a pellet and a
supernatant
Subcellular centrifugation - a way to separate the
cell contents based on density of organelles
Cytosol - not an organelle but a result of
centrifugation

Differential centrifugation

Use of density of organells to isolate cell

fractions
C ytosolic
fraction

600 g
x 10 min

Homogenate

15,000 g
x 5 min

Nuc lear
fraction

105,000 g
x 60 min

Mitochondrial
fraction

Microsomal
fraction

Density gradient centrifugation

Also called Zonal centrifugation - Performed in the presence of an
increasing dense solution
often sucrose or other materials (percol most common)
can be used to purify a specific organelle or determine the
sedimentation and ultimately the molecular weight of a protein

Ammonium sulfate precipitation

salting out proteins
At high concentrations of this strong salt, water is highly ordered
High concentration of strong chaotropic salts strips water away
from protein
Lower availability of solvent (water)
This favors protein interactions rather than protein - solvent
interactions causes aggregation of proteins (they become
insoluble)
Each protein has a
different solubility so
this is a method to
isolate groups of
protein
Precipitation is reversible
and usually non
damaging to structure
of the enzyme
Ammonium Sulfate is
most commonly used.
Urea is also used but is
usually is harder for the

Column Chromatography

Separation based the interactions between a

mobile phase and the chromatographic media
(stationary phase)
Used to separate any of the big four biomolecules

Column Chromatography

Separation based the interactions between a

mobile phase and the chromatographic media
(stationary phase)
Used to separate any of the big four biomolecules
Components
Column
Pump
Absorbance monitor
Conductivity monitor
Fraction collector
Controller - for more
advanced work
(control freaks?)

Affinity chromatography

purification based on a natural interactions for a

protein and a substrate or chemical group
(ligand)
only proteins which recognize the molecule on the
stationary phase will bind
Elute by competition with the bound ligand
generally a good method but it doesnt always work
- Some non-specific interactions can occur
Spacer arm may be needed to make the
compound available to the protein

Examples of ligand - protein affinity matrix

ATP. Glutathione, nickel small molecules attached

to a ligand
Fusion proteins can take advantage of affinity by acting as a

tag:

glutathione S transferase (GST) binds to glutathione

histidine6 - binds to a nickel column

Power of biochemistry and molecular biology an

example of affinity chromatography

Ras - small protein involved in several cancers

Low concentration in cells, so it difficult to purify and study
create a fusion protein 1/2 Ras 1/2 Glutathione S-transferase

(GST) and produce large amounts of it.

lead to discovery of additional proteins involved in Ras
regulation

Ion exchange chromatography

- separation of proteins based on net charge of protein exchange of ions for proteins
Anion Exchanger
weak exchanger - diethylaminoethyl (DEAE)
strong exchanger - quatenaryaminoethyl (QAE)
This type of resin is positively charged
The resin binds negative proteins
Proteins are eluted by NaCl or altering pH - how does this
work?

Cation
exchanger
weak exchanger
- carboxymethy
(CM)
weak exchanger
- sulfipropyl (SP)
protein eluted
by the same
means as above

Size exclusion (SEC) or gel filtration chromatography

Media (solid phase) is a defined pore sizes in polymer beads,
large molecules go around small molecules go through and around the
beads
Smaller sized proteins are retained and come out last
Range of types of beads and chemistry - resin can be made of agarose,
acrylamide or other polymers

Size exclusion (SEC) or gel filtration chromatography

Can be used to separate proteins, remove salts exchange buffers or to
determine the molecular weight of a purified protein
Also used to determine the molecular weight of a protein - use protein
standards with known molecular weights, prepare a standard curve of these
known proteins and compare the elution volumes of the knowns to the
unknowns

Example
Sephacryl S-200 has a fractionation range of 5
kDa to 250 kDa
?What is the exclusion limit
Would this be appropriate for a set of proteins
with molecular weights of 8 kDa, 15 kDa, 200
?kDa and 500 kDa
What about 15, 250, 310, 405 kDa
? if you wanted the 15 kDa protein
What about if you wanted to purify the 310
?kDa protein

Protein Characterization
Electrophoresis - The transport
of particles by an electrical
field through a solid media

a good method for

determining the purity of a
protein and analyze a mixture
of proteins

Separation of charged
compounds based on an
applied electrical field, net
charge and frictional coefficient
(mass and shape of molecule)

Similar to DNA gels

proteins and very small DNA (oligonucleotides) use acrylamide

PAGE (Polyacrylamide
Gel Electrophoresis)
the Gel is a
polymerized
Acrylamide- alter the ratio

and concentration of polymer and

crosslinker to alter the pore size
and change the

migration through the gel

A low % gel (acrylamide) will
separate higher MW proteins while
smaller proteins are not well
resolved
Stacking Gel vs. Resolving Gel- need to get the proteins to start
at the same time (compressing the
proteins into a

narrow starting zone at the

(resolving gel

Denatured Electrophoresis - SDS PAGE

Separation of proteins based on size not charge

Add a reducing agent - -mercaptoethanol or

dithiothreitol
and a detergent - sodium dodecyl sulfate (SDS)
then boil to unravel the protein and solvate protein with
ampipathic SDS

- each SDS has 2 negative charges

- many SDS per molecule
- total amount of SDS bound is proportional to the

MW

- Each protein molecule will be sufficiently

negative
Therefore each protein will be very negativity charge
regardless of the amino acid composition,
- The size of protein influences the migration separation is based on size only not charge.

Native Gel
Electrophoresis

Separations based on native

size and charge
Two proteins of a similar size
but different charge will
migrate differently
Protein interactions can
influence the migration of
protein

Isoelectric Focusing Electrophoresis

Separation of proteins based on isoelectric point
Native or denatured electrophoresis in a pH gradient of
polyampholytes
pH gradient is formed when electrical field is applie
Proteins will migrate, depending on net charge, until there is no longer
a charge on the protein.
How does this happen?

2 Dimensional Electrophoresis
Combination of native or denatured PAGE and IEF
Run in two directions
1- PAGE - to separate by size
2- IEF to separate by charge alone
Good to separate very crude mixtures or determine
the difference between two proteins that are the same size
but with a different pI

2D-Electrophoresis
2D-electrophoresis allows separation of proteins
by both size and isoelectric point. Each spot
represents a different protein. The horizontal
represents the isoelectric focusing direction,
while the veritcal represents the SDS PAGE
direction.

Immuno Analysis

Immunoglobins - 5 major classes main antibody

response in sera is IgG
antigen - foreign substance that triggers antibody
formation
epitope - section of antigen that antibody recognizes

Antibodies consist of heavy and

light chains
Fab region - highly variable recognize target (antigen)
FC heavy chain - interacts with
other proteins
polyclonal vs. monoclonal
antibodies

polyclonal
from sera of an animal
several epitopes to the same antigen
some may cross react with other proteins in a nonspecific manner
produce lots of antibodies al long as the animal lives
and you continue to boost

monoclonal

derived from single cell - hybrid of mouse spleen and a immortal cell line
(lymphocyte and myeloma)
inject mice then can grow cell in a dish
antibodies purified from cell culture media
single epitope, very specific
unlimited production of antibodies

Antibodies in specific analysis

ELISA (Enzyme Linked ImmunoAssay)- most

sensitive detection methods for antibodies (aids
test), proteins, peptides and other substances
(drug testing)

Plastic Dish

Antibodies in specific analysis

ELISA (Enzyme Linked ImmunoAssay)- most
sensitive detection methods for antibodies (aids
test), proteins, peptides and other substances
(drug testing)

Plastic Dish

Protein of interest is
Bound to plastic

Antibodies in specific analysis

ELISA (Enzyme Linked ImmunoAssay)- most
sensitive detection methods for antibodies (aids
test), proteins, peptides and other substances
(drug testing)

2 Unreacted binding sites

are Covered with a nonreactive protein

Plastic Dish

Protein of interest is
Bound to plastic

Antibodies in specific analysis

ELISA (Enzyme Linked ImmunoAssay)- most
sensitive detection methods for antibodies (aids
test), proteins, peptides and other substances
(drug testing)

2 Unreacted binding sites

are Covered with a nonreactive protein

3 Primary Antibody

Recognizes Antigen

Plastic Dish

Protein of interest is
Bound to plastic

Antibodies in specific analysis

ELISA (Enzyme Linked ImmunoAssay)- most
sensitive detection methods for antibodies (aids
test), proteins, peptides and other substances
(drug testing)

Secondary Antibody
Conjugated to an enzyme

2 Unreacted binding sites

are Covered with a nonreactive protein

3 Primary Antibody

Recognizes Antigen

Plastic Dish

Protein of interest is
Bound to plastic

Antibodies in specific analysis

ELISA (Enzyme Linked ImmunoAssay)- most
sensitive detection methods for antibodies (aids
test), proteins, peptides and other substances
(drug testing)

5 Enzyme reacts
with substrate
producing colored
product

Secondary Antibody
Conjugated to an enzyme

2 Unreacted binding sites

are Covered with a nonreactive protein

3 Primary Antibody

Recognizes Antigen

Plastic Dish

Protein of interest is
Bound to plastic

Western blot - good for

mixtures of proteins,
identifying size and
characteristics
transfer proteins form
SDS PAGE to paper for
antibody analysis.
Primary antibody
recognizes protein
antigen
a secondary antibody
recognizes the Fc region
and is conjugated to a
second molecule to act as
a signal