FINAL - CH 5 Flashcards
Protein Purification Factors
- pH
- Temperature
- Presence of degradative enzymes
- Adsorption to surfaces
- Long-term storage
Order of fractions in centrifugation
Nuclear
Mitochondrial
Membrane
Cytosol
Soilbility
Salting out
Ionic charge
ionic exchange chromotography
Electrophoresis
Isoelectric focusing
Polarity
Hydrophobic interaction chromotography
Size
Gel filtration chromotography
SDS-PAGE
Binding specificity
Affinity chromotography
Chromatography Techniques
Gel filtration
Ion-Exchange
Affinity
High Performance Liquid Chromatography
version of gel filtration
leads to greater separation of proteins that are a similar size
High pressure is required to force buffer and protein through the column
pH = pI
No interaction will occur between protein and column
pH > pI
-
pH < pI
+
Cation exchanger
- charged column
CMC
Anion exchanger
+ charged column
DEAE
Specific activity
total units of activity / total protein
Multifold increased purity
Specific activity of sample in question/Specific activity of sample before it
Total yield***
total units of sample in question/Total units of step 1
Total multifold purification
Specific activity in question/Specific activity in step 1
Technique for the separation and visualization of
proteins based on the migration of charged
proteins in an electric field
Electrophoresis
PAGE
Polyacrylamide gel electrophoresis
Separates proteins on the basis of charge and size
percentage of gel in PAGE
low % - small molecules migrate faster, better separation between large molecules
High% - separation better between small molecules
SDS-PAGE
detergent that assists molecules to the anode (+)
denatures proteins
Coomassie Brilliant Blue G-250
used to stain proteins in PAGE
Isoelectric focusing
Separates proteins based on isoelectric point
Proteins will stop migrating once they reach the pH that matches their PI
Two Dimensional (2D) Gel Electrophoresis
Isoelectric focusing combined with SDS-PAGE
…………………………………High mass
………………………………….Low Mass
Low PI…………..High PI
2D Differential In-Gel Electrophoresis
Uses covalently bound fluorescent dyes (Cy3 and Cy5) to
distinguish two proteins run on the same SDS
PAGE gel
Edman degradation can sequence an oligopeptide up to ____ amino acid residues
50
Edman degradation
PITC is covalently attached to the N-terminal amino acid and then treated with TFA which cleaves between the first and second amino acid
For peptides LARGER than 50 AAs
enzymatic cleavage with trypsin and
chymotrypsin is performed
chymotrypsin
Cleaves at C-side of Tyr, Trp, and Phe
trypsin
Cleaves on the C-side of Lys and Arg
Cyanogen bromide
Cleaves on C-side of Met
V8 protease
Cleaves on the C-side of Asp and Glu
Mass Spectrometry
Measures the mass of small peptide
fragments
mass-to-charge ratio (m/z)
Peptide Ionization Methods
(mass spec)
Electrospray ionization
Matrix-assisted laser desorption ionization
Electrospray ionization
(mass spec)
Solution of peptide is sprayed
high voltage forms 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
Tryptic fragments are embedded in a light-absorbing
matrix
Fragments are released as charge molecules after laser
exposure
A detector determines the mass
DNA sequencing
Faster than sequencing AA
can be used only if Gene has been isolated
Solid Phase Peptide Synthesis
generate oligopeptides of up to 25 amino acids
Protein Structure Determination
X-ray crystallography
NMR spec
X-ray Crystallography two steps
Growing diffraction quality crystals
Determining the phases of the diffracted X-rays
Process of X-ray Crystallography
Xray interacts with electrons
X-ray structure is an image of the electron density of the object
Computer graphics assist in the structure determination
better ordered crystals
Have higher resolution, Lesser resolution limit
lesser ordered crystals
Have lower resolution, Higher resolution limit
Crystalline proteins assume _________ structures that they have in solution
nearly the same
Because crystals are hydrated,
Crystalline protein is in solution, which allows is to be present in normal form
NMR spec
Used to determine the relative locations of atoms in a purified protein solution
NMR vs Xray
Validates X-ray data
Can determine structures of proteins that fail to crystallize
More dynamic
Antibody proteins are produced by _____ in
the immune system.
B cells
The immune response is triggered by the
presence of a foreign macromolecule, often a
protein or carbohydrate, known as an ______
antigen
Each B cell makes a _____ type of antibody.
single
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)
the most common immunoglobulin
IgG
Heavy chain: γ
Light chain: λ or κ
IgG sub unit structure
γ2κ2 OR γ2λ2
Epitope
Specific site on antigen that can bind to the antibody
Each antigen is surrounded by _____ different antibodies
4
Polyclonal antibodies
Heterogeneous mixture of immunoglobulin proteins that recognize one or more epitopes on an antigenic protein
Monoclonal
Homogeneous immunoglobulin species that recognizes one epitope on an antigenic protein
Solid step synthesis 5 steps
Deblocking of residue 1 attached to resin
Activation of Fmoc-blocked residue 2
coupling of AA
Final deblocking
Cleavage and deprotection
Generation of Polyclonal Antibodies
Immunize rabbit 4 times of 6 week period
obtain blood sample
Blood will contain non specific and specific antigens
Purification of antigen specific antibodies using affinity chromotography
Generation of Monoclonal Antibodies
Multiple immunizations of lab animal
Isolate B cells
Fuse B cells and tumor cells
both antigen specific and non specific hybridomas will grow
isolate antigen specific by screening
Clone to increase amount
Western Blotting uses 2 antibodies
Primary (protein-specific)
Secondary (detection antibody)
Western blotting is used to:
detect proteins separated by gel
electrophoresis
antibody-based technique used to identify
proteins in cells that have been chemically
treated in a way that preserves cell
architecture
Immunofluorescence
antibodies are used to identify proteins that are associated with protein antigens by combining mass spec
Immunoprecipitation
