6052009
BICHBICH
605; Fall
October 6, 8, 20 & 22
Larry Dangott
Department of Biochemistry and Biophysics
Room 440 BioBio
845-2965
ljdangott@tamu.edu
BICH
605
OUTLINE
Planning:
 Method Development; Strategies
 Activity Tracking; Fraction ‘pooling’
Techniques:
 Electrophoresis (SDS, Isoelectric Focusing)
 Chromatography (GFC, IEX, Affinity, rpHPLC)
 Structural Characterization (Amino Acid Analysis; Protein
Sequencing)
 Proteomics (Protein ID and characterization using mass
spectrometry)
To present an OVERVIEW of techniques used in Protein
Purification and Analysis.
Protein Purification
It helps to know something about your protein
1. Source (organism; tissue; organelle;
amount)
2. Assemblage vs. monomer
3. Cytosolic vs. membrane-bound
4. Size
5. Isoelectric point (pI)
6. Post-translational modification
7. Relative abundance
Protein Purification
Source (organism; tissue; organelle; amount)
1. Natural vs. Recombinant (tagged?)
2. Tissue (bone (hard), blood (liquid), heart
(soft), brain (fatty)); extraction
3. Organelle (nucleus, mitochondria, ER,
plasma membrane); pre-fractionation
4. Amount (a LOT or a little; scale); cost and
practicality (myoglobin = easy; EGFR =
hard)
Protein Purification
Multimer vs. Monomer
Affects buffer choices (assembly vs. disassembly)
Affects choice of separation media (size)
Cytosol vs. Membrane
Affects pre-fractionation choices (extraction)
Separation methods (centrifuge, columns)
Affects buffer choices (detergent)
Size (sort of related to Multimer vs. Monomer)
Affects choice of separation media (GFC)
Affects solubility (larger proteins like to precipitate)
Isoelectric point (pI)
Affects choice of separation media (charge)
Affects solubility (precipitate at pI)
Affects buffer choices (precipitation point; charge)
Post-Translational Modification
Affects choice of separation media (affinity)
Protein Purification
Important Steps You May Use:
• Extraction (French press,
sonication, detergent,
homogenization)
• Centrifugation (low speed, ultraspeeds, differential gradient)
Protein estimation method
(colorimetric, spectroscopy)
• Protein concentrating method
(salt or organic precipitation,
lyophilization, membrane
filtration)
• Chromatography (IEX, gel
filtration, chromatofocusing)
• Electrophoresis (IEF, preparative
native or SDS)
Protein Purification
A COMPLEX STRATEGY FOR PROTEIN PURIFICATION
Sample Preparation
•
•
Extraction (grinding, detergent lysis, sonication)
Salt exchange (gel filtration, filters, dialysis)
Capture
•
•
•
Ion Exchange
Affinity
Hydrophobic Interaction
Intermediate Purification
•
•
Ion exchange
Hydrophobic Interaction
Polishing
1. Gel Filtration
2. Reversed phase
A SIMPLE STRATEGY
His-tag: affinity
Protein Purification
Systematic method development requires.....
Defining a way of quantifying, or at least identifying, the
presence of your target molecule, and of assessing its purity.
Don’t rely solely on literature (or coworker) statements. Verify
yourself. 50% success rate.
Keep a record of your purification process.
Notebook, notebook, notebook………. .
.
.
Happy Boss
Protein Purification
Our Example: Enzyme Purification
There are two major objectives in enzyme
purification:
To obtain the highest SPECIFIC ACTIVITY possible, measured
as activity per unit protein
To
obtain
the
MAXIMUM
YIELD
of
enzymatic
activity.
(Theoretically, this is 100%. Practically, one is usually happy to
settle for something like 30%.)
Protein Purification
When purifying a protein, one wants to keep track
of how one is doing relative to the two major
objectives.
Therefore, at each step, one must measure:
1. Volume
2. Protein concentration (colorimetric assay, UV)
3. Enzyme activity (units/ml; specific to ‘your’
protein)
Protein Purification
These measurements are combined in the calculation of:
Total activity = Enzyme activity/aliquot volume X Total volume
Total protein = Protein/aliquot volume X Total volume
Specific activity = Enzyme activity in an aliquot/Amt of Protein in
the aliquot (THIS IS THE BIG ONE)
(In measurements of total activity and protein, remember to adjust
for volumes set aside for various reasons. If this is not done, the
yield will be artificially low).
Calculate Activity Units and Total Protein
Use to calculate Specific Activity
Vol X Activity Units/vol = Total Activity Units
Vol X mg/ml = Total Protein (mg)
Divide Total Activity Units by Total Protein (mg) = Specific Activity in Units/protein (mg)
Fold purification goes UP
Yield goes DOWN
Divide current Specific Activity by Initial Specific Activity = Fold Purified
Divide current Total Activity Units by Original Activity Units = % Yield
KNOWING WHICH FRACTIONS TO POOL IS IMPORTANT
Selecting Fractions based on Specific Activity and SDS PAGE
Mutant Tyrosine Hydroxylase; Ion Exchange; NaCl Gradient
RNA?
Pool
Stable?
Pure Mutant TyrOH has a Vmax of ~12
Data courtesy of Colette Daubner; Fitzpatrick Lab
Protein Purification
The less prevalent the protein is in the cytosol, the
higher the degree of purification that will be
required for its purification to homogeneity.
For example:
A protein that is 50% of the cellular protein needs to
be purified only 2-fold.
In contrast:
A protein that is only 0.1% of the cellular protein
needs to be purified 1000-fold.
Protein Purification
A TYPICAL STRATEGY FOR PROTEIN PURIFICATION
1.
Sample Preparation
1.
2.
2.
Capture
1.
2.
3.
3.
Ion Exchange
Affinity
Hydrophobic Interaction
Intermediate Purification
1.
2.
4.
Extraction (grinding, detergent lysis, sonication)
Salt exchange (gel filtration, filters, dialysis)
Ion exchange
Hydrophobic Interaction
Polishing
1.
2.
Gel Filtration
Reversed phase
Mode of monitoring the purification………………
Protein Purification
Tracking your protein is critically important. How do you
know where it is?
Biological Assay (usually specific; extremely sensitive; slower)
Binding Assay (usually specific; sensitive, semi-automate)
Chemical Assay (colorimetric assays, enzyme assays)
Physical Assay (mass spec, UV spectrometry)
Separation Assay (electrophoresis)
SDS PAGE ELECTROPHORETIC ANALYSIS OF
PROTEINS
Electrophoresis is a electrically driven sieving process used to separate complex
mixtures of proteins. Can be ANALYTICAL or PREPARATIVE.
SDS PAGE is used to investigate subunit composition and to verify homogeneity of
protein samples. It can also serve to purify proteins for use in further microanalytical applications
Principle of SDS PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)
Most proteins bind the ionic detergent, SDS (sodium dodecyl sulfate), in a constant
weight-to-detergent ratio, leading to identical negative charge density per mass for
the denatured proteins and a uniform shape.
Thus, theoretically, SDS-protein complexes migrate through a solid matrix
(polyacrylamide) and are separated according to size, not charge.
SDS PAGE
APPLICATIONS
Polypeptide composition and fraction profiling:
Purified protein complexes or multimeric proteins consisting of
subunits of different molecular size will be resolved into
constituent polypeptides. Screen fractions during protein
purification.
Quaternary structure profile:
Comparison of the protein bands obtained under non-reducing
and reducing conditions provides information about the
molecular size of subunits and protein complexes.
Size estimation:
The relationship between the relative mobility and log molecular
weight is linear over some range. With the use of plots like those
shown here, the molecular weight of an unknown protein (or its'
subunits) may be determined by comparison with known protein
standards.
SDS PAGE
In SDS gel electrophoresis, negatively charged, SDS-coated proteins migrate
in response to an electrical field through pores in a crosslinked
polyacrylamide gel matrix
Pore size decreases with higher acrylamide concentrations
Smaller pores are used for smaller proteins/peptides; larger pore sizes are
used for larger proteins.
SDS PAGE
PROCEDURE
Proteins to be analyzed are
solubilized and denatured by
boiling (or heating) in the
presence of SDS and reducing
reagent, an aliquot of the protein
solution is applied to a gel lane,
and the individual proteins are
separated electrophoretically.
The reducing reagent βMercaptoethanol (-ME) or
(dithiothreitol (DTT)) is added
during solubilization to reduce
disulfide bonds.
SDS PAGE
The polyacrylamide gel is cast as a
separating gel (sometimes called the
resolving or running gel) topped by a
stacking gel and secured in an
electrophoresis apparatus (see
figure).
The stacking gel is run at slightly acid pH
(6.8). The separating gel is run at pH
8.8. The stacking gel is ~4%
acrylamide and the separating gel is
a higher concentration.
The stacker brings the proteins to a common ‘starting line’ and the separator
sieves them apart. The concentration of acrylamide in the separating gel is
determined by the range of molecular weights of interest.
SDS PAGE
Tris-Glycine in Upper Buffer
Tris-HCl pH 6.8 in Stacking Gel
Tris-HCl pH 8.8 in Separating Gel
SDS PAGE
Glycine equilibria
SDS PAGE
Formation of an ion front
SDS PAGE
It is the voltage gradient that sharpens the ion boundary
SDS PAGE
What happens to proteins?
SDS PAGE
In separating gel
Glycine mobility increases, becomes greater than protein mobility, but still
slower than Cl-
SDS PAGE
Protein sample, now in a narrow band, encounters
both the increase in pH and decrease in pore size.
Increase in pH would tend to increase electrophoretic
mobility, but smaller pores decrease mobility.
Relative rate of movement of ions in separating gel is
chloride > glycinate > protein.
Proteins separate based on charge/mass ratio and on
size and shape parameters.
SDS PAGE
PROTEIN DETECTION
Detection limit
Fixing?
Coomassie Blue G-250 or R-250 staining
50 ng
fixing
Silver
1 ng
fixing
10 ng
non-fixing
1- 10 ng
non-fixing
Fluorescent stains
(Sypro)
Negative stains (zinc, copper)
Coomassie Blue
Silver
Sypro Ruby
SDS PAGE
SIZE ESTIMATION
IMPORTANT
MW ESTIMATION BY SDS-PAGE
IS ONLY APPROXIMATE AND IS
REFERRED TO AS APPARENT
MOLECULAR WEIGHT. Unusual
protein compositions or physical
properties can cause anomalous
mobilities during SDS-PAGE.
SDS gels can be used as a micropurification step and the individual
polypeptides can be isolated from
the gel by electroelution or
electroblotting and the amino acid
sequences can be determined or
peptide maps obtained.
Relative Mobility (Rf)
ISOELECTRIC FOCUSING
IEF is a technique to separate proteins
based on Isoelectric Point (native or denatured)
Isoelectric Point (pI) is specific pH at which
net charge equals zero
At pI, protein has no net charge
and will not migrate in an electric
field
Isoelectric Focusing
IEF CAN BE PERFORMED WITH MOBILE pH GRADIENTS OR IMMOBILIZED pH
GRADIENTS
Mobile gradients are prepared with Carrier
Ampholytes (CAs) (mixed polymers (300-1000
Da in size) mixed with solid support (mobile).
Immobile gradients are prepared by covalently
coupling Ampholytes to solid support and
blending.
Solid support is usually polyacrylamide but can
be agarose for preparative purposes
Isoelectric Focusing
MOBILE pH GRADIENT IEF
IMMOBILIZED pH GRADIENT IEF
ADVANTAGES of
IMMOBILIZED GRADIENTS
•Stable pH gradients
•Ease of handling
•Reproducibility
•Extreme pH resolution
2 Dimensional Gel Electrophoresis
Combine IEF & SDS PAGE
High Resolution
Zoom gels (pH range)
Detect isoforms
Post-translational modifications
Expression Proteomics
2 Dimensional Gel Electrophoresis
Combine IEF & SDS PAGE
•High Resolution
•Zoom gels (pH range)
ANALYTICAL
Detect isoforms
Post-translational modifications
PREPARATIVE
Mass spectrometry
END OF DAY 1