Chapter Outline

 5.1 The Art and Science of Protein

Purification
 5.2 Working with Oligopeptides: Sequencing
and Synthesis
 5.3 Protein Structure Determination
 5.4 Protein-Specific Antibodies Are Versatile
Biochemical Reagents
Proteomics
 High throughput protein biochemical methods
to study the proteome
 Provides insight into how changes in an
organism’s physiology alters the
proteome
5.1 The Art and Science of Protein
Purification
 Generally uses freshly isolated cells or
tissues
 Relies on exploiting the inherent
qualitative and quantitative differences in
biochemical properties of individual
proteins
 Uses specific biochemical assays to
uniquely identify proteins of interest among
all other proteins contained within a sample
to be analyzed
 Generally detect a product of a chemical reaction
performed by the protein of interest
Luciferase: An Early Biochemical
Assay
 An enzyme found
in bioluminescent
organisms
 Uses luciferin as
a substrate
Luciferase Assay and Consequence
Cell Fractionation: Preparation
 Cells are broken open and cell extracts (homogenates)
are produced.
 Protein activity is retained.

 Cell suspensions are prepared by mincing tissue

mechanically or enzymatically to generate membrane-
bound cells that can be homogenized in one of three
ways:
 Sonication
 Shearing (with French press)
 Incubation with mild detergents

 Centrifugation is performed to increase the concentration

and purity of the target protein in the sample.
Cell Fractionation
Cell Fractionation: Fraction
Formation
 Fractions are created.
 Each fraction contains less total protein
than the beginning of the cell extract which
enriches the target protein.
 This enrichment can be expressed as
specific activity
 Specific activity = the total amount or activity of
the target protein divided by the total amount of
protein in the fraction.
Preparative Centrifugation
Post Centrifugation: Salting Out
 Used to exploit differences in solubility of
the target protein relative to other proteins in
the fraction
 Involves adding increasing amounts of
saturated salt solutions to the protein sample
 This causes the formation of insoluble
protein aggregates that are functional when
resolubilized.
Dialysis
 Used to remove
ammonium
sulfate from the
protein sample
 Uses diffusion to
leave protein in
the buffer the
proper ionic
strength and pH
Chromatography Techniques
 Column

 Gel filtration

 Affinity
Column Chromatography, Part 1
 Used to separate proteins based on different
physical or chemical interactions between
the proteins and the column matrix
 Uses a column to separate individual proteins
from a sample
 Separated proteins (fractions) can
be collected in small containers
Column Chromatography, Part 2
Column Chromatography, Part 3
33
Click to view the Column Chromatography
animation
Gel Filtration Chromatography

 Separates proteins
based on size
 Also called size-
exclusion
chromatography
 Large proteins migrate
faster than smaller
proteins, which may
get trapped in the
carbohydrate beads.
High Performance Liquid Chromatography
(HPLC)
 High-resolution version of gravity-based
gel filtration chromatography
 Column matrix contains smaller particle
beads and leads to greater separation
of proteins that are a similar size.
 High pressure is required to force buffer and
protein through the column.
Ion-Exchange
Chromatography,
Part 1
 Exploits charge
differences between
proteins
 Uses two matrices:
 Positively charged, anion-
exchange matrix (DEAE)
 Negatively charged, cation
exchange matrix (CMC)
Ion-Exchange Chromatography, Part 2
Affinity Chromatography, Part 1
 Exploits specific binding properties of the
target protein to separate it from other
cellular proteins that lack this function
 High-affinity ligand for the target protein is
covalently linked to the matrix bead.
 Other proteins pass through the column.
 An antibody column can also be used to
isolate antigenic proteins.
Affinity Chromatography, Part 2
Purification Efficiency of a
Target Protein
Gel Electrophoresis
 Used to approximate the molecular mass of
a protein or if the purified protein includes
more than one polypeptide chain
 Polyacrylamide gel electrophoresis (PAGE)
 Separates proteins on the basis of charge and
size
 Percentage of gel is important depending on
protein size
PAGE
Diagram
SDS-PAGE
 PAGE using sodium dodecyl sulfate (SDS), a detergent
that adds a net negative charge to the protein to aid in
migration to the anode
 This is used to denature the proteins.
SDS Gel Electrophoresis
Click to view the SDS Gel Electrophoresis
animation
Protein Separation
Protein Staining after Separation
Isoelectric Focusing
 Separates proteins based on isoelectric point
Two Dimensional (2D) Gel
Electrophoresis
 Isoelectric focusing combined with
SDS- PAGE
 Separates proteins based on pI and
molecular mass
 Can separate thousands of proteins
into discrete spots
2D Gel Electrophoresis
2D Differential In-Gel
Electrophoresis (DIGE)
 Uses fluorescent dyes (Cy3 and Cy5) to
distinguish two proteins run on the same
SD PAGE gel
 Proteins are covalently labeled with different
fluorescent dyes.
DIGE
Schematic
DIGE Gel
5.1 Working with Oligopeptides:
Sequencing and Synthesis
 The work of Frederick Sanger
Edman Degradation, Part1
 Improved Sanger’s protein sequencing
method
 Phenylisothiocynate (PITC) is covalently
attached to the N-terminal amino acid
and then treated with TFA.
 The polypeptide is cleaved between the
first and second amino acid.
 This is an automated process that can
sequence an oligopeptide up to 50
amino acid residues.
Edman
Degradation, Part 2
Protein Cleavage, Part 1
 For proteins longer than 50 residues,
enzymatic cleavage with trypsin and
chymotrypsin is performed.
 Trypsin
 Cleaves on the C-side of Lys and Arg
 Chymotrypsin
 Cleaves at C-side of Tyr, Trp, and Phe
Protein Cleavage, Part 2
 Cyanogen bromide
 Cleaves on C-side of Met
 S. aureus
 Cleaves on the C-side of Asp and Glu
Protease
Cleavage
Mass Spectrometry
 Measures the mass of small peptide
fragments
 Measures the mass-to-charge ratio (m/z)
 Can be used to determine the molecular
mass
Peptide Ionization Methods
 Electrospray ionization (ESI)
 Releases polypeptides that have been cleaved by
trypsin
 Matrix-assisted laser desorption ionization
(MALDI)
 Tryptic fragments are embedded in a light-
absorbing matrix.
 Fragments are released as charge molecules
after laser exposure.
 A detector determines the mass.
Mass Spectrometer Diagram
Solid Phase Peptide Synthesis,
Part 1
 Used to synthesize peptide antigens
for antibody production
Solid Phase Peptide
Synthesis, Part 2
5.3 Protein Structure Determination

 Two primary methods for determining

molecular structure:
 X-ray crystallography
 Nuclear magnetic resonance spectroscopy
X-ray Crystallography, Part 1
 Based on the diffraction of X-rays by protein
crystals
 1957 – Structure of myoglobin
was determined.
 Two steps:
 Growing diffraction quality crystals
 Determining the phases of the diffracted X-rays
X-ray
Crystallography, Part 2
X-ray Crystallography for
Nitrophorin 2
NMR Spectroscopy
 Exploits the magnetic properties of several
types of atoms
 Specifically 1H, 15N, and 13
C
 Nuclear spins are aligned.
 Only works for proteins less than 100 kDa
5.4 Protein-Specific Antibodies Are
Versatile Biochemical Reagents
 Antibody proteins are produced by B cells
in the immune system.
 Each B cell makes a single type of antibody.
 B cells can make:
 Two classes of Ig light chains (λ and κ)
 Five classes of Ig heavy chains (µ, α, δ, ε, and γ)
Immunoglobulins
 Contain several domains
 Variable (VL and VH)
 Constant (CL and CH1, CH2, and CH3)
 Fab fragment contains the antigen binding site.
 Epitope
 Specific site on antigen that can bind to the antibody
Polyclonal vs. Monoclonal Antibodies
 Polyclonal  Monoclonal
 Heterogeneous  Homogeneous
mixture of immunoglobulin
immunoglobulin species that
proteins that recognizes one
recognize one or epitope on an
more epitopes on an antigenic protein
antigenic protein
Generation of Polyclonal Antibodies
Generation of Monoclonal Antibodies
Western Blotting
 Used to detect proteins separated by gel
electrophoresis
 Uses two antibodies:
 Primary (protein-specific)
 Secondary (detection antibody)
Western
Blotting
Schematic
Epitope Tagging
 Protein-coding sequences of highly
antigenic peptides are added to protein-
coding sequences of cloned genes.
Immunofluorescence, Part 1
 An antibody-based technique used to
identify proteins in cells that have been
 chemically treated in a way that preserves
cell architecture
 After washing, cells can be visualized
directly through fluorescence microscopy if
the primary antibody is fluorescent or the
secondary antibody contains a fluorescent
group.
Immunofluorescence, Part 2
Enzyme Linked Immunosorbent
Assay (ELISA)
 Identifies low
level
antigenic
proteins
 Typically used
in biological
samples
Immunoprecipitation
 Variation of affinity
purification
 Monoclonal antibody
is covalently linked to
a carbohydrate bead.

 Co-immunoprecipitation
can be performed as
well.