Chapter 5 - Methods in Protein Biochem Flashcards
If pH > PI:
Molecule is negative and will bind to positively charged medium
Called an anion exchanger
If pH < PI:
Molecule is positive and will bind to negatively charged media
Called a cation exchanger
1,000 g for 5 minutes
Supernatant: Mitochondrial, membrane, cytosol
Pellet: Nuclear fraction
10,000 g for 10 minutes
Supernatant: Membrane, cytosol
Pellet: Mitochondrial
15,000 g for 15 minutes
Supernatant: Cytosol
Pellet: Membrane
extracting a protein bound to a column by a solvent that removes the protein from the stationary phase to the mobile phase
Elution
Generally, the chemistry of Fmoc blocking is straightforward for most amino acids during solid state peptide synthesis. There is one amino acid, however, that presents a problem for Fmoc blocking during solid state peptide synthesis. That amino acid is
Lysine
It has an R-NH3+ group that will react with Fmoc and thus this side chain group also needs blocking
this method uses an X-ray beam aimed at a homogeneous protein crystal in solid phase.
X-ray crystallography
Provides information on the structure of a purified protein based on its nuclear spin properties in a magnetic field
NMR Spectroscopy
Use of high-affinity antibodies linked to a dense carbohydrate bead to isolate protein antigens
Immunoprecipitation
Low-percentage gel
Increases rate of protein migration
Separates larger proteins at expense of smaller protein separation
High-percentage gel
Decreases rate of protein migration
Separates smaller proteins at expense of larger protein separation
2-D gels:
_________________Larger
_________________Smaller
Low PI High PI
_______ cleaves on the carboxy side of aromatic amino acids
Chymotrypsin
W, Y, F
_______ cleaves Lysine
Trypsin
Luciferase
An enzyme found in bioluminescent
organisms
Uses luciferin as a substrate
If you add luciferin substrate to different protein samples:
The protein sample that illuminates contains luciferase
membrane bound cells can be homogenized in one of three ways:
Sonication
Shearing (with French press)
Incubation with mild detergents
Centrifugation
Performed to increase the concentration
and purity of the target protein in the sample
Cell suspensions are prepared by
mincing tissue mechanically or enzymatically to generate membrane bound cells
Specific activity
The total amount or activity of the target protein divided by the total amount of protein in the fraction
How to find the magnitude of purification
Multiply the specific activities of cell extract (un purified sample) and cytosol fraction
Protein Purification Factors
pH Temperature Presence of degradative enzymes Adsorption to surfaces Long-term storage
Protein characteristic: Solubility
Salting out
Protein characteristic: Ionic charge
Ionic exchange solubility
Electrophoresis
Isoelectric focusing
Protein characteristic: Polarity
Hydrophobic interaction chromatography
Protein characteristic: Size
Gel filtration chromatography
SDS-page
Protein characteristic: Binding specificity
Affinity chromatography
Salting out
causes the formation of insoluble protein
aggregates that are functional when
resolubilized
Process of salting out
a) Salt is added to a concentration just below the precipitation point of the target protein
b) After centrifugation, unwanted proteins are discarded and more salt is added to salt out the target protein
c) After a second centrifugation, the protein is recovered as a precipitate.
As you add more salt:
Salt and ions are competing for solvent
Salt wins staying in solution forcing other proteins into pellet
Dialysis
Used to remove ammonium sulfate from protein sample
Gel Filtration Chromatography separates proteins based on:
Size
In Gel Filtration Chromatography:
Large travel faster than small proteins
High Performance Liquid Chromatography (HPLC)
Leads to greater separation of proteins similar in size
Uses pressure to push buffer and molecules into column
Positively charged, anion exchange matrix
(DEAE)
Negatively charged, cation exchange matrix
(CMC)
Ion exchange chromatography
Separates molecules on the basis of differences in
their net surface charge
Affinity Chromatography
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
________ to the matrix bead.
covalently linked
The binding between the ligand and target
molecule must be _____ to allow target
molecules to be ______ in an active form.
reversible
removed
The specific activity is a measure of ______: it increases during purification of an enzyme and becomes _______ when the enzyme is pure.
enzyme purity
maximal and constant
Technique for the separation and visualization of
proteins based on the migration of charged
proteins in an electric field
Electrophoresis
Electrophoresis is carried out:
In gels made up of the cross-linked
polymer polyacrylamide
PAGE
Polyacrylamide gel electrophoresis
Separates proteins on the basis of charge and size
Percentage of gel is important depending on protein
size
SDS-PAGE
Uses sodium dodecyl sulfate, a detergent
that adds a net negative charge to the protein to aid in
migration to the anode
Used to denature the proteins
Migration from _____ to _____
Cathode (-)
Anode (+)
Staining of proteins in electrophoresis:
Coomassie Brilliant Blue G-250
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
2D Differential In-Gel Electrophoresis (DIGE)
Proteins are covalently labeled with different
fluorescent dyes
Uses Cy3 (green) and Cy5 (red)
Nobel prize in Chemistry for protein sequencing
Frederick Sanger
Improved Sanger’s protein sequencing
method
Edman degradation
Edman degradation:
________ (PITC) is covalently
attached to:
Phenylisothiocynate
the N-terminal amino acid andthen treated with TFA
Edman degradation can
sequence an oligopeptide up to:
50 amino acid residues
For proteins longer than 50 AA:
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
Cyanogen bromide:
Cleaves on C-side of Met
S. aureus:
Cleaves on the C-side of Asp and Glu
Sanger reaction can determine ______ in protein sequencing
N-terminus AA
Protein Sequencing: How to determine the composition
- Purify the protein of interest – separate away from all
other types of proteins and biomolecules - Estimate the molecular weight of the protein
- Establish the composition by complete hydrolysis of the protein under acidic conditions
Protein Sequencing: How to determine the order
- Determine the C-terminal amino acid
- Identify the N-terminal amino acid
- Cleave disulfide bonds between and within polypeptides
- Cleave peptide bond using proteases in a specific way.
- Determine the aa sequences through repeated cycles of Edman degradation.
- Conduct a second round of protein cleavage and compare the aa sequences of the overlapping sets of peptide fragments.
- Finally determine the positions (if any) of the disulfide bonds.
How to determine the C-terminal amino acid
Use carboxypeptidase – enzyme that removes the last (C-terminal) amino acid in a free form by breaking the peptide bond
Hydrolyzes the peptide bond nearest the C-terminus
How to identify the N-terminal amino acid
Use 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride)-fluorescent compound that reacts with primary amines
How to cleave disulfide bonds between and within polypeptides
Use 2-mercaptoethanol or another mercaptan
Treat with iodoacetate to prevent the reformation of disulfide bonds through oxidation by O2
Cleave peptide bond using proteases in a specific way
Trypsin – Cleaves on the C-terminal side of Lys and Arg residues
Chymotrypsin – Cleaves on the C-terminal side of Tyr, Phe, and Trp
Chemical proteases also can cleave proteins: Cyanogen Bromide (CNBr) – cleaves on the C-terminal side of Met
How to determine the aa sequences through repeated cycles of Edman degradation.
If the sequence of each peptide is determined, the entire protein sequence can be reassembled from the fragments
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)
Matrix-assisted laser desorption ionization (MALDI)
Electrospray ionization (ESI)
Solution of peptide is sprayed from a narrow capillary tube maintained at high voltage (~4000 V), forming fine, highly charged droplets from which solvent rapidly evaporates.
Dry N2 gas promotes the evaporation
The charges result from the protonation of Arg and Lys.
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.
When the gene is isolated, sequencing the DNA can be:
Faster and more accurate than sequencing the protein.
Most proteins are now sequenced in this indirect way:
DNA Sequencing
If the gene has not been isolated, direct sequencing of _____ is necessary.
peptides
Amino acid sequence provides information about protein structure that is not revealed by DNA sequencing:
Location of disulfide bonds
Solid Phase Peptide Synthesis
a method to generate oligopeptides of up to
25 amino acids.
Solid Phase Peptide Synthesis involves adding ______ to the peptide through a ______ on the amino terminus
one amino acid at a time
covalent linkage
Used to synthesize peptide antigens for antibody
production and to manufacture peptide-based
therapeutic drugs to treat a variety of diseases.
Solid Phase Peptide Synthesis
Solid Phase Peptide Synthesis Step 1
De-blocking of residue 1 attached to resin
Solid Phase Peptide Synthesis Step 2
Activation of Fmoc-blocked residue 2
Solid Phase Peptide Synthesis Step 3
Coupling of AAs
Solid Phase Peptide Synthesis Step 4
Final de-blocking of peptide with 20 AAs
Solid Phase Peptide Synthesis Step 5
Cleavage form resin and de-protection of all amino side chains
Two primary methods for determining molecular
structure:
X-ray crystallography - use crystal of pure protein
NMR Spectroscopy - measures magnetic characteristics of each atom
X-ray crystallography
Based on the diffraction of X-rays by protein
crystals
X-ray crystallography 2 steps:
Growing diffraction quality crystals
Determining the phases of the diffracted X-rays
laboratory X-ray generators
Synchrotrons
X-ray crystallography determined structure of ____
Myoglobin
Protein crystals are highly _______
hydrated
40-60% water by volume
Protein crystals typically have resolution limits in the
range:
1.5 to 3.0 Å
Better ordered crystals have _____ resolution and _____ resolution limit
higher, lesser
Less ordered crystals have _____ resolution and _____ resolution limit
lower, higher
Many ____ are catalytically _____ in the crystalline
state.
enzymes
active
Crystal packing forces ____ greatly perturb the
______ of protein molecules.
do not
structures
Protein in a crystal is essentially ______ because it is
bathed by _______ over its surface
except for the few
in solution
solvent of crystallization
NMR Spec only works for proteins less than:
100 kDa
NMR Spec Validates:
X-ray data
NMR spec can determine:
Structures of proteins that fail to crystallize
NMR spec resolution:
2-2.5 Å resolution
NMR Spectroscopy
Used to determine the relative locations of atoms in a purified protein solution
Antigen
a foreign macromolecule, often a protein or carbohydrate, that triggers the immune system
Antibody proteins are produced by ____ in
the immune system.
B cells
B cells can make:
Two classes of Ig (immunoglobulin) light chains (λ and κ)
Five classes of Ig heavy chains (µ, α, δ, ε, and γ)
IgG
the most common immunoglobulin, is equally distributed between the blood and the extravascular fluid.
IgG structure:
Light chain: λ or κ
Heavy chain: γ
Subunit structure: γ2λ 2 OR γ2κ2
Molecular mass: 150 kD
The association between
antibodies and their antigens
involves:
van der Waals, hydrophobic, hydrogen bonding, and ionic
interactions.
Antigen-antibody complex have:
high specificity and strength
The immune system has the potential to produce
an enormous number of different antibodies
> 10^18
The diversity in antibodies results from
gene recombination and mutation
Polyclonal antibodies
Heterogeneous mixture of immunoglobulin proteins
recognize one or more epitopes on an antigenic protein
Monoclonal antibodies
Homogeneous immunoglobulin species
recognizes one epitope on an antigenic protein
Western blotting
Used to detect proteins separated by gel electrophoresis
Western blotting uses two antibodies:
Primary (protein-specific)
Secondary (detection antibody)
Protein-coding sequences of highly antigenic
peptides are added to protein-coding
sequences of cloned genes.
Epitope Tagging
An antibody-based technique used to identify
proteins in cells that have been chemically
treated in a way that preserves cell
architecture
Immunofluorescence
Enzyme Linked Immunosorbent
Assay (ELISA)
Identifies low level antigenic proteins
Typically used in biological samples
The amount of substrate converted indicates the amount of protein present
Immunoprecipitation
Variation of affinity purification
In in mmunoprecipitation _______ is ______ linked to a carbohydrate bead.
monoclonal antibody
covalently
In immunoprecipitation the ________ combination is
used to _______ from other proteins physically using _________
antibody-bead
separate protein antigens
low speed centrifugation
In immunoprecipitation antibodies can be used to identify proteins that are associated with __________________ by combining _____________
protein antigens in large cellular complexes
immunoprecipitation with mass spectrometry
addition of a phosphate group makes the protein more:
negatively charged
chelation affinity chromatography
purifies recombinant proteins with 6 or more histidine residues which have strong affinity for divalent metals
Antibody column
isolates antigenic proteins in the sample
Epitope tag
protein-coding sequences of highly antigenic peptides
ESI
electrospray ionization (mass spec)
MALDI
matrix-assisted laser desorption/ionization (mass spec)
ESI uses
high voltage
generates highly charged molecule in gas phase
MALDI uses
laser
proteins are fragmented and charged
Primary antibody
facilitate antigen-antibody interaction
Secondary antibody
recognizes primary antibody, assists in detection
Each B cell makes how many antibody type
One
How many classes of immunoglobulin heavy chain are produced
5
How many classes of immunoglobulin light chain are produced
2
Secondary antibodies are typically from:
A different animal as the primary antibody
