Download GMS BI 555/755 Lecture 3: Techniques for

GMS BI 555/755 Lecture 3: Techniques for Protein
Characterization
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Reading: Berg/Stryer, 6th Ed. Chapter 3
Protein extraction, sub-cellular fractionation
Size-based separations
– Dialysis
– Gel filtration
– Ultracentrifugation
Charged-based separations
– Ion Exchange chromatography
Affinity chromatography
Reversed phase chromatography
Electrophoresis
– SDS-PAGE
– Isoelectric focusing
– 2D Gel electrophoresis
Circular dichroism
Amino acid analysis
Edman degradation
Peptide synthesis
Proteomics
Sept. 16, 2009
GMS BI 555/755 Lecture 3.
Protein: from Greek πρώτα
("prota"), meaning "of
primary importance"
Protean: readily assuming
different forms or
characters; extremely
variable
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Proteins must be extracted from their cell or tissue context
for purification
Classical and contemporary reasons
for sub-cellular fractionation
•Yield, activity, specific activity
• Differential Centrifugation. Cells are disrupted in
a homogenizer and the resulting mixture, called
the homogenate, is centrifuged in a step-bystep fashion of increasing centrifugal force. The
denser material will form a pellet at lower
centrifugal force than will the less-dense
material. The isolated fractions can be used for
further purification.
• Alternatives: cells may be disrupted by
treatment with low or high salt (osmotic lysis)
and the protein in question purified from the
cytosol or membrane fraction.
• Selective precipitation according to protein
class
• Ammonium sulfate ppt
• NaCl ppt
• Ethanol precipitation of ECM proteins
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GMS BI 555/755 Lecture 3.
•Enzyme activity
•Patterns of protein expression
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Variable properties:
•Solubility
•Size
•Charge
•Hydrophobicity
•Binding/adhesion
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Size based separations: dialysis
Centrifugal concentrators:
• Centrifugal force pushes solution
through a membrane with size
selective pores
• Useful for concentrating, desalting
proteins
• Also dialysis concentrator cells
Dialysis. Protein molecules (red) are
retained within the dialysis bag,
whereas small molecules (blue) diffuse
into the surrounding medium.
• Separation of large from small
molecules
• Solubility, recovery serious issues
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Size based separations: Gel-filtration chromatography
• AKA size exclusion chromatography
• Smaller proteins experience a higher
mobile phase volume because they are
able to enter pores of the stationary
phase beads.
• The elution volume is inversely related to
the molecular weight
• Chromatographic resolution depends on a
number of factor including bead diameter,
pore size, salt concentration, column
volume, flow rate
• Other factors constant, resolution
increases with column volume
• Protein must be freely soluble
• Detergents necessary for membrane
proteins
• Typically 0.15 M salt necessary to prevent
non-specific interactions between proteins
and beads.
• Volume of protein injected onto column
must be <2% of column volume.
• Eluant is diluted
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Gel filtration/Size exclusion
Gel filtration: principles and
methods, GE Healthcare
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Gel filtration/Size exclusion
Gel filtration: principles and
methods, GE Healthcare
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Size based separations: Gel-filtration chromatography
Gel filtration chromatography of a protein mixture
(1) thyroglobulin (669 kd),
(2) catalase (232 kd),
(3) bovine serum albumin (67 kd),
(4) ovalbumin (43 kd), and (
(5) ribonuclease (13.4 kd).
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Gel filtration/Size exclusion: sample volume
Gel filtration: principles and
methods, GE Healthcare
5/23/2017
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Log scale
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SEC: optimization of flow rate
Van Deemter equation
A = Eddy-diffusion
B = Longitudinal diffusion
C = mass transfer kinetics of the analyte
between mobile and stationary phase
u = Linear Velocity.
48 Da
500 Da
1000 Da
Ziegler, A.; Zaia, J. J Chromatogr B Analyt Technol Biomed Life Sci 2006, 837, 76-86.
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Protein analysis by ultracentrifugation
Sub-cellular fractionation (preparative)
Analysis of protein mass, density, shape, binding (analytical)
A particle will move through a liquid medium
when subjected to a centrifugal force. A
convenient means of quantifying the rate of
movement is to calculate the sedimentation
coefficient, s, of a particle by using the following
equation:
where
m is the mass of the particle,
 is the partial specific volume (the reciprocal of
the particle density),
ρ is the density of the medium and
f is the frictional coefficient (a measure of the
shape of the particle).
The (1 - ρ) term is the buoyant force exerted by
liquid medium.
Sedimentation coefficients are usually expressed
in Svedberg units (S), equal to 10-13 s. The
smaller the S value, the slower a molecule
moves in a centrifugal field.
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Protein
S value
(Svedberg
units)
Molecular
weight
Pancreatic
trypsin inhibitor
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Cytochrome c
1.83
12,310
Ribonuclease A
1.78
13,690
Myoglobin
1.97
17,800
Trypsin
2.5
23,200
Carbonic
anhydrase
3.23
28,800
Concanavlin A
3.8
51,260
Malate
dehydrogenase
5.76
74,900
Lactate
dehydrogenase
7.54
146,200
6,520
From T. Creighton, Proteins, 2nd Edition (W. H.
Freeman and Company, 1993), Table 7.1.
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Protein analysis by ultracentrifugation
1. The sedimentation velocity of a particle depends in
part on its mass. A more massive particle sediments
more rapidly than does a less massive particle of the
same shape and density.
2. Shape, too, influences the sedimentation velocity
because it affects the viscous drag. The frictional
coefficient f of a compact particle is smaller than that of
an extended particle of the same mass. Hence,
elongated particles sediment more slowly than do
spherical ones of the same mass.
3. A dense particle moves more rapidly than does a less
dense one because the opposing buoyant force (1 - ρ) is
smaller for the denser particle.
4. The sedimentation velocity also depends on the
density of the solution. (ρ). Particles sink when ρ < 1,
float when ρ > 1, and do not move when ρ = 1.
Density and Sedimentation Coefficients of Cellular Components. [After L. J.
Kleinsmith and V. M. Kish, Principles of Cell and Molecular Biology, 2d ed. (Harper
Collins, 1995), p. 138.]
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Protein analysis by ultracentrifugation
5% sucrose
25% sucrose
Zonal Centrifugation. The steps are as follows: (A) form a density gradient, (B) layer the sample on
top of the gradient, (C) place the tube in a swinging-bucket rotor and centrifuge it, and (D) collect the
samples. [After D. Freifelder, Physical Biochemistry, 2d ed. (W. H. Freeman and Company, 1982), p.
397.]
•Protein mass can be measured accurately by sedimentation equilibrium
•Mass measurement under native conditions, applicable to multiprotein complexes
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Charge-based separations: ion exchange chromatography
Weak Cation exchange (WCX) Weak Anion exchange (WAX)
Strong cation exchange (SCX)
Sulfonic acid functional groups
Strong anion exchange (SAX)
Quaternary amino ethyl (QAE)
• A protein in a buffer at pH > pKa will be negatively charged and able to bind
an anion exchange resin.
• A protein in a buffer at pH < pKa will be positively charged and able to bind
a cation exchange resin
• Proteins may be eluted with a gradient of increasing salt (NaCl, or other)
• The elution order will depend on how tightly (charged) is a given protein
• Injection volume not limited
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Charge-based separations: ion exchange chromatography
• Ion exchange entails loading a protein mixture at a given pH in a low salt concentration buffer and
washing the unbound material through the column
• For cation exchange, positively charged proteins bind to the column, negatively charged proteins
pass through
• For anion exchange, negatively charged proteins bind the column, positively charged ones pass
through
• Bound proteins are eluted by a gradient of increasing salt concentration
• Weakly bound proteins elute at a relatively low salt concentration, tightly bound ones at higher salt
concentration
• Proteins are focused by binding to the column. Thus, large volumes of solution may be applied to
the column.
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Affinity Chromatography
• A protein may be purified based
on its binding to a chemical group
or another protein
• Immobilize the ligand on a
chromatographic bead (many
standard chemistries available)
• Pass the protein mixture through
the column
• Wash non-bound material through
the column
• Elute the bound proteins either
using a competing ligand, an
increasing salt concentration, or
other denaturatn
• Examples
• Avidin-biotin
• Poly-His tags binding to
immobilized metal columns
• Protein A/G binding to
antibodies
• Growth factor binding to
heparin columns
• Transcription factor binding
to immobilized DNA seq
• Purification of recombinant
fusion proteins (GST, GFP,
FLAG)
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High performance liquid chromatography
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Chromatographic resolution increases as
the size of the chromatographic beads
decreases
The pressure required to push solvent
(mobile phase) through the packed beads
increases as their size decreases
High performance liquid chromatography
(HPLC): a pumping system that pushes
solvent through a column packed with small
beads (approximately 5 microns).
All tubing, columns, made of stainless steel
to withstand pressures up to 2500 psi or so.
Higher performance and cost than
conventional low pressure liquid
chromatography.
Typically one begins with low pressure
chromatography to concentrate the protein
and then switches to HPLC for later steps
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•Available with any chromatography mode
•Many models and manufacturers
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% acetonitrile
• Reversed phase HPLC is a
ubiquitously useful means of
separating proteolytic peptides
using a gradient from low to high
percent organic
• Peptides bind to C18 stationary
phase in low organic solvent, elute
with a gradient of increasing
organic content
• Trifluoroacetic acid (TFA) is an
ion-pairing agent that prevents
charge-based interactions
between peptides and stationary
phase
• Proteins are denatured by TFA
and organic
• Useful as a means for separating
intact proteins for structural
studies.
Absorbance 214 nm
Reversed phase high performance liquid chromatography
Time
Reversed phase separation of a tryptic digestion of
apotransferrin. Gradient of 0-50% acetonitrile, 0.1%
trifluoroacetic acid over 100 min,
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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE)
protein purity, homogeneity, distribution
Thin gel, molecular sieve
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Polyanionic
~1 SDS/2AA
• Proteins prepared by boiling in an SDSbuffer in the presence of a reductant
(mercaptoethanol, DTT)
• Protein solution loaded into a gel lane
• Applied electric field causes protein-SDS
complexes to migrate to the anode
(positively charged) electrode at the bottom
• Proteins separated approximately based on
size as they migrate through the porous
polyacrylamide gel matrix
GMS BI 555/755 Lecture 3.
ν =Ez/f
ν = velocity
E = elec. field str.
z = net prot charge
f = frictional coeff. 20
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE)
Chromatographic fractions
Staining of Proteins After Electrophoresis.
Proteins subjected to electrophoresis on an
SDS-polyacrylamide gel can be visualized by
staining with Coomassie blue.
•Formation of cross-linked polyacrylamide gel.
•Many recipes for pouring gradient gels for protein separations
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Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE)
Log scale
•Migration proportional to log(mass)
•Can resolve 2% difference in mass
•Resolution = m/Δm = ~50
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GMS BI 555/755 Lecture 3.
• Protein-SDS complexes migrate
approximately according to mass
• Mobility proportional to the logarithm
of mass
• This assumes that the ratio of bound
SDS molecules per mass unit of
protein is the same for all proteins
• In reality, this ratio, and thus the
relative mobility in the electric field,
varies according to several factors
• Hydrophobicity of polypeptide
(membrane proteins migrate
differently than soluble
proteins)
• Glycosylation: most proteins
are glycosylated, and many
glycans are acidic
• Phosphorylation: ubiquitous
signalling mechanism, lowers
pI
• Mature protein heterogeneity
results in diffuse bands.
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Cytochrome C pI 10.8
Serum albumin pI 4.8
Two-dimensional gel electrophoresis
IEF
• Isoelectric point = pI
= pH where z = 0
• Loading an issue
The Principle of Isoelectric Focusing. A pH
gradient is established in a gel before loading the
sample. (A) The sample is loaded and voltage is
applied. The proteins will migrate to their isoelectric
pH, the location at which they have no net charge.
(B) The proteins form bands that can be excised
and used for further experimentation.
Two-Dimensional Gel Electrophoresis. (A) A protein sample
is initially fractionated in one dimension by isoelectric focusing.
The isoelectric focusing gel is then attached to an SDSpolyacrylamide gel, and electrophoresis is performed in the
second dimension, perpendicular to the original separation.
Proteins with the same pI are now separated on the basis of
mass. (B) Proteins from E. coli were separated by twodimensional gel electrophoresis, resolving more than a
thousand different proteins. The proteins were first separated
according to their isoelectric pH in the horizontal direction and
then by their apparent mass in the vertical direction.
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>1000 spots stained
i.d. a problem
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2D electrophoresis
Advantages
• Separation and visualization of 1000’s
of proteins in two dimensions based on
pI and molecular weight
• Able to visualize protein pattern easily
• Comparison of protein expression
patterns as a function of biological
change
• Many commercial 2DE systems
available, chemistries well worked out
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Disadvantages
• Dynamic range (limited protein
loading)
• Visualizes the most abundant proteins;
low abundance proteins not detected
• Proteins must be excised from gels for
identification using Edman degradation
or mass spectrometry
• Difficult to get reproducible separation;
alignment of different gels requires
sophisticated software
• Most animal proteins are posttranslationally modified and are
observed as more than one spot.
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Circular dichroism and protein structrure
Ranjbar, B.; Gill, P. Chem Biol Drug Des 2009, 74, 101-120.
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Circular dichroism for protein structure analysis:
characteristic profiles according to secondary structure.
Lesk: Introduction to Protein Science, chap 3, Fig. 11
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CD: fixed wavelength measurement for determination of protein
melting point (protein stability)
Ranjbar, B.; Gill, P. Chem Biol Drug Des 2009, 74, 101-120.
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Use of CD to assess the folded state of a protein:
• A properly folded protein has CD signal in both near and far UV
regions
• A molten globule protein has secondary, not tertiary structure, and
lacks near-UV signal
Ranjbar, B.; Gill, P. Chem Biol Drug Des 2009, 74, 101-120.
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Amino acid analysis
•Is the protein pure?
•Is it the right protein?
•Does it have the expected AA composition?
nm
Amino acid analysis involves four basic steps:
1. Hydrolyze a protein to individual constituent amino acids (6N HCl)
2. Label amino acids with a detectable UV-absorbing or fluorescent marker
3. Separate different types of amino acids by chromatography
4. Measure relative amounts of each amino acid type based on intensity of the detectable marker
associated with the emergence of each type of amino acid from the chromatographic system
Requires a few hundred picomoles of purified protein
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Amino acid analysis
Fluorescamine reacts with the α-amino
group of an amino acid to form a
fluorescent derivative.
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Ala-GlyAsp-Phe-Arg-Gly
Determination of Amino Acid Composition. Different amino
acids in a peptide hydrolysate can be separated by ionexchange chromatography on a sulfonated polystyrene resin
(such as Dowex-50). Buffers (in this case, sodium citrate) of
increasing pH are used to elute the amino acids from the
column. The amount of each amino acid present is
determined from the absorbance. Aspartate, which has an
acidic side chain, is first to emerge, whereas arginine, which
has a basic side chain, is the last. The original peptide is
revealed to be composed of one aspartate, one alanine, one
phenylalanine, one arginine, and two glycine residues.
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Amino acid analysis
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Useful method for establishing that a given protein preparation has an
amino acid composition that matches its theoretical sequence
Used in industry for recombinant protein-based products
– Recombinant drugs
– Antibodies
– Animal feeds and additives
Requires a relatively large quantity of pure protein, primarily due to
limitations of the hydrolysis step (high temperature, 6N HCl)
~300 pmol or 30 μg of a 100 kDa protein
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Edman Degradation: N-terminal protein sequence analysis
Pehr Edman, 1950, automated protein sequencer, 1967
• Phenylisothiocyanate (PITC) reacts with protein N-terminus.
Under mild acid conditions to form a phenylthiohydantoin (PTH)
amino acid derivative
• The reaction may be repeated, one cycle per AA residue
• PTH-AAs detected using chromatography
• Much engineering and optimization of reaction and detection
system
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Edman Degradation
The labeled amino-terminal residue (PTH-alanine in the first round) can be released without hydrolyzing
the rest of the peptide. Hence, the amino-terminal residue of the shortened peptide (Gly-Asp-Phe-Arg-Gly)
can be determined in the second round. Three more rounds of the Edman degradation reveal the complete
sequence of the original peptide.
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Edman Degradation
Separation of PTH-Amino Acids. PTH-amino acids can be rapidly separated by high-pressure
liquid chromatography (HPLC). In this HPLC profile, a mixture of PTH-amino acids is clearly
resolved into its components. An unknown amino acid can be identified by its elution position
relative to the known ones.
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Edman Degradation
Advantages:
• Relatively straight-forward
interpretation based on
chromatographic retention time
• Automated gas phase Edman
sequencers available (?)
• Able to handle 2 or 3 component
mixtures in favorable cases
• Absolute quantification of released
amino acids
• Can analyze PTM-modified AAs
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Disadvantages
• Slow, ~1 hr per cycle
• Peptides must be pure, not able to
handle complex mixtures
• Blocked N-terminus prevents Edman
degradation (Acetylation, pyroglutamic
acid, others)
• Used primarily for analysis of proteinbased pharmaceutical products
• Heroic effort necessary to completely
sequence a protein
• Lack of market for commercial
instruments
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Cleavage of proteins at specific amino acid residues:
Peptide mapping
• Met is a rare AA residue
• Cyanogen bromide cleavage
generates large peptides
(membrane proteins)
Cleavage by Cyanogen Bromide. Cyanogen bromide cleaves
polypeptides on the carboxyl side of methionine residues
Cleavage by Trypsin. Trypsin hydrolyzes polypeptides on the
carboxyl side of arginine and lysine residues
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GMS BI 555/755 Lecture 3.
• Lys and Arg are relatively common AA
residues
• Trypsin digestion reliably cleaves to the
C-terminal side for denatured proteins
• Produces peptides ranging
approximately from 500-2500 Da (many
exceptions)
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Reliable methods for specific protein cleavage
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Enzymatic
– Trypsin (C-term of Lys, Arg)
– Lys-C (C-term of Lys)
– Asp-N (N-term of Asp)
– Chymotrypsin (Tyr, Trp, Phe, Leu, Met, others)
– (Other enzymes less reliable)
Chemical
– Cyanogen bromide
– (Other chemical methods less reliable)
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Informational value of amino acid sequences
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BLAST (Basic Local Alignment Search Tool) www.ncbi.nih.gov computes
alignments for a given sequence to known sequences
Does a protein belong to a given family?
Evolutionary trees based on protein sequence similarity
Are there internal repeats in a protein sequence? Evidence for duplication
of primordial genes
Sequence data may be used to generate antibodies against a protein of
interest (Western blotting, immunohistochemistry, immunoprecipitation,
ELISA)
AA sequences useful for design of DNA probes specific to the
corresponding gene.
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Solid phase peptide synthesis
Amino Acid Activation. Dicyclohexylcarbodiimide is used
to activate carboxyl groups for the formation of peptide
bonds.
• Solid phase peptide synthesis starts by immobilizing the
C-terminal amino acid of the target peptide sequence on a
resin
• The t-Boc protecting group is removed
• The second t-Boc amino acid is then added and coupled
using carbodiimide chemistry
• Carbodiimide reacts with and activates the carboxyl group
of the t-Boc-AA to facilitate peptide bond formation
Berg
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Solid phase peptide synthesis
The first amino acid (blue) of the desired
peptide is attached at its carboxyl end by
esterification to a polystyrene bead. The amino
group of this amino acid is blocked by the
attachment of a tertbutyloxycarbonyl (tBOC)
group (red), which is removed by treatment with
trifluoroacetic acid (CF3COOH). The resulting
free amino group forms a peptide bond with a
second amino acid, which is presented with a
reactive carboxyl group and a blocked amino
group, together with the coupling agent
dicyclohexylcarbodiimide (DCC). The process
is repeated until the desired product is
obtained; the peptide is then chemically
cleaved from the bead with hydrofluoric acid
(HF). [See R. B. Merrifield, L. D. Vizioli, and H.
G. Boman, 1982, Biochemistry 21:5020.
Lodish
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ELISA: Enzyme-linked Immunosorbent Assay
Immobilization: physical or chemical?
Indirect ELISA and Sandwich ELISA (A) In indirect ELISA, the production of color indicates the amount of
an antibody to a specific antigen. (B) In sandwich ELISA, the production of color indicates the quantity of
antigen. [After R. A. Goldsby, T. J. Kindt, B. A. Osborne, Kuby Immunology, 4th ed. (W. H. Freeman and
Company, 2000), p. 162.]
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ELISA
Advantages
• Extremely sensitive detection of Ab or
Ag in question
• Amenable to high-throughput multiwell
format
• Quantitative readout of Ag or Ab level
in a fluid
• Many fluorescent tags available for
ELISA detectionn (radiolabeling)
• Many commercial kits for ELISA
development, detection, etc.
Sept. 16, 2009
Disadvantages
• Requires high quality Abs to each Ag
to be tested; time consuming and
expensive to generate Abs (requires
high affinity, specific Abs)
• Generation of high quality Abs is an
inexact science
• ELISA conditions (buffer salt
concentrations, additives, detergents,
washing protocolls, etc) are specific to
each Ag-Ab interaction; it is difficult to
make a single assay for more than one
Ag-Ab pair.
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Western Blotting
Western Blotting. Proteins on an SDS-polyacrylamide gel are transferred to a polymer sheet and stained
with radioactive (or fluorescently labeled) antibody. A band corresponding to the protein to which the
antibody binds appears in the autoradiogram.
• Able to detect presence of a given protein in a complex cell lysate
• Semi-quantitative measurement of protein level
• Depends on transfer of proteins from gel to membrane
• Not all abs have high enough affinity to be useful for Western blotting
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Genomics and Proteomics
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Genomics: organismal study of patterns of gene expression related to
disease and developmental processes
– Human genome project: all human genes sequenced
– Approximately 30k human genes
Functional genomics: effort to make use of the vast wealth of data from the
various genomics projects to understand gene and protein functions and
interactions
– Focusses on dynamic aspects of gene transcription, translation, and
protein-protein interaction
Proteomics: large scale study of protein expression: functions and
structures
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Why study protein expression?
(the steps of gene expression control)
Nucleus
Cytosol
RNA
degradation
control
DNA
primary
RNA
transcript
mRNA
inactive
mRNA
mRNA
translation
control
transcriptional
control
RNA
processing
control
RNA
transport
control
Consequence: protein expression is usually poorly correlated to
mRNA expression level
protein
modified
protein
posttranslational
control
Gygi, et al., Mol. Cell. Biol., 1999, 19, p. 1720-1730)
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Some common post-translational modifications of proteins
Mann, M., and Jensen, O. N. (2003). Nat Biotechnol 21, 255-61.
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