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Lecture 2
1. Extraction of proteins
San-Yuan Huang
Lab. Animal Proteomics
Dept. of Animal Science, NCHU
Mach 6, 2008
Chromatographic methods
Size exclusion chromatography
Ion exchange chromatography
Affinity chromatography
Metal binding
Immunoaffinity chromatography
High performance liquid chromatography (HPLC)
(http://en.wikipedia.org/wiki/Protein_purification)
Introduction
In order to isolate intracellular proteins, cells must be
disrupted.
How is the protein to be used?
Is the protein required for peptide sequencing in order
to perform cloning and sequencing the cDNA?
Is the protein required for investigation of its properties
and biological activities?
Strategies and considerations are required in choosing
the right condition for protein extraction
Theory of buffering
In order to ensure reproducible experimental results, it is important to
maintain the protein solution at the constant pH.
Buffering: partially neutralized solutions of weak acid or weak
bases are resistant to pH changes on the addition of
small amounts of strong acid or strong base.
HA (acid) H++A- (incomplete)
NaANa++ A- (complete)
(1.1)
(1.2)
The addition of small amounts of strong acid (H+) to the buffer shifts
equilibrium (Equation 1.1) to the left using A- supplied by Equation 1.2.
Whereas the addition of small amounts of strong base (OH-) combines with
H+ provided by equilibrium (Equation 1.1) moving to the left.
In either case, change of H+ concentration, hence pH, is unchanged.
Examples
For in gel permeation chromatography, almost any buffer suitable
for the protein of interest can be chosen.
anion-exchange chromatography, cationic buffers such as Tris (and
for cation-exchange chromatography, anionic buffers such as
phosphate) are preferred. However, these inorganic buffers do
have some side effects.
Phosphate buffers are shown to inhibit many enzymes including
carboxypeptidase, urease, kinase, and dehydrogenase.
Examples
Tris and other primary amine buffers may form Schiff base adducts
with aldehydes and ketones and inhibit protein conjugation by
amine-based cross-linkers.
Borate buffers may form covalent complexes with the ribose
moieties of nucleic acids, and other mono- and oligosaccharides.
However, the Good buffers (developed by Good et al.) such as
MES, PIPES, and MOPS have been shown to be relatively free of
side effects, low UV absorbance and are minimally affected by
temperature or ionic strength, useful with respect to low metal
binding capabilities, retaining most metals essential to enzymatic
activity.
Preparation of buffers
In practice, the buffer is usually prepared at room
temperature and the pH adjusted so that it provides the
correct pH after the solution is brought to the desired
temperature.
The pH of the working buffer should be tested after all
the components (e.g., ethylenediamine tetraacetic acid
[EDTA], dithiothreitol [DTT], Mg2+) have been added,
since the pH may change after such additions.
Unless otherwise stated, the pH of a buffer is adjusted
down with hydrochloric acid (HCl) and up with either
NaOH or KOH.
Effect of temperature on pH
The carboxylic acid buffers (of low pKa) are generally least
sensitive to temperature.
The amine buffers (of high pKa) have temperature-sensitive pKa.
For example, pKa of Tris changes from 8.85 at 0 to 8.06 at
25.
dlnKa/dT=H0/RT2
or
dpKa/dT=H0/2.3RT2
and
G0=H0- TS0=-RTlnKa =2.3RTpKa
Effect of concentration on pH
It is a common practice to prepare buffers as 10x to
100x stocks.
This allows smaller storage volumes and further dilutes
the bactericidal agents such as 0.02% sodium azide to
an insignificant level before use.
It is important to note that the dilution of the stock buffer
solution may change the pH.
For example, the pH of Tris decreases by 0.1 unit per
tenfold dilution, while a tenfold dilution of 0.1 M
phosphate buffer (pH 6.7) raises the pH to 6.9.
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Volatile buffers
Volatile buffers are used when it is necessary to
remove the buffer quickly and completely.
Volatile buffers are particularly useful in the
digestion of proteins followed by separation of
peptides or amino acids.
Most volatile buffers are compatible with
ninhydrin.
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Ionic detergents
Contain head groups with either positive (cationic
detergents) or negative charges (anionic
detergents).
Ionic detergents denature proteins; thus, these are
not included in the extraction.
Sodium dodecyl sulfate (SDS) and
cetyltrimethylammonium bromide (CTAB) are
examples of anionic and cationic detergents,
respectively.
Non-ionic detergents
Have uncharged hydrophilic head groups and have a
poly-oxyethylene polar region.
Generally used to isolate functional proteins, as they
are far less denaturing than ionic detergents.
Common examples of non-ionic detergents are Triton
X-100 and Tween 20.
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Zwitterionic detergents
Contain head groups with both positive and negative
charges, bile salts or steroid-based detergents.
These detergents are more effective than non-ionic
detergents, as they prevent protein-protein interactions
while causing less protein denaturation than ionic
detergents.
CHAPS is the most commonly used zwitterionic
detergent.
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The CMC is defined as the lowest detergent concentration at which micelles form.
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Detergent
conc.
very low
1:10
1:1
10:1 to
20:1
Detergent extraction of a
membrane protein
occurs as follows:
a. binding of detergent
to the membrane
and initiation of lysis
b. solubilization of the
membrane in the
form of detergentlipid-protein complex
c. further solubilization
of the detergentlipid-protein
complexes to give
detergent-protein
complexes and
detergent-lipid
complexes.
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Consideration of pH
Carboxylic acid-containing detergents, such as bile salts and Nlauryl sarcosinate, may not be suitable in some experiments related
to separation techniques (isoelectric focusing, pH gradient elution
from ion-exchange resin, chromatofocusing) where pH values vary.
Detergents of this class are expected to protonate and become
insoluble in aqueous media at weakly acid pH value.
Zwitterionic detergents containing stronger acids such as sulfonic
acid and sulfonate ester have pK values 0.2 and thus do not present
such problems.
Consideration of temperature
Detergents containing polyoxyethylene ethers such as Triton X-100
and Triton X-114 change micelle molecular weight with temperature.
The micelle expands in an exponential fashion as the temperature
increases. This process leads to a separation of detergent as a nonaqueous phase at a particular temperature, known as the cloud point,
this temperature effect is advantageous for the extraction of
membrane proteins but not in the electrophoretic techniques, such as
isoelectric focusing, where heat is generated.
Other non-ionic or zwitterionic detergents may be appropriate for such
techniques.
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Homogenization
Homogenization in a blender and in a Dounce are
common and simple procedures form disruption soft
tissues such as liver, heart, brain, and muscle.
These methods are rapid (5 to 10 min) and gentle to
proteins.
These homogenization procedures usually produce
heat, and thus the blender and the associated
container should be prechilled at 4.
The homogenization should be performed m a cold
room or on ice.
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Sonication
Sonication is most commonly used to disrupt various
types of cells (prokaryotes and eukaryotes).
Sonication creates vibrations that cause mechanical
shearing of the cell wall.
Sonication is performed at the highest allowable power
setting, which is adjusted to a level slightly below that
at which foaming occurs.
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Dispersions
Degassing
Homogenization
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procedures.
Refolding of the denatured protein in the thiol reagents and by
dilution or dialysis.
SDS is also an effective solubilizing agents
difficult to remove from most proteins
interfere with the subsequent refolding
incompatible with ion-exchange chromatography.
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