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Purification and
Characterization
Protein Purification and
Characterization
Why Study proteins?
Cyt osolic
fraction
Components
– Column
– Pump
– Absorbance monitor
– Conductivity monitor
– Fraction collector
– Controller - for more
advanced work
(control freaks?)
Affinity chromatography
purification based on a natural interactions for a protein and a substrate
or chemical group (ligand)
– only proteins which recognize the molecule on the stationary phase
will bind
– Elute by competition with the bound ligand
– generally a good method but it doesn’t always work - Some non-
specific interactions can occur
– Spacer arm may be needed to make the
compound available to the protein
Examples of ligand - protein
affinity matrix
ATP. Glutathione, nickel – small molecules attached to a ligand
Fusion proteins can take advantage of affinity by acting
as a tag:
– glutathione S transferase (GST) –binds to glutathione
– histidine6 - binds to a nickel column
Power of biochemistry and molecular biology an example of affinity
chromatography
– Ras - small protein involved in several cancers
Low concentration in cells, so it difficult to purify and
study
create a fusion protein 1/2 Ras 1/2 Glutathione S-
transferase (GST) and produce large amounts of it.
lead to discovery of additional proteins involved in Ras
regulation
Ion exchange chromatography
- separation of proteins based on net charge of protein - exchange of
ions for proteins
Anion Exchanger
weak exchanger - diethylaminoethyl (DEAE)
strong exchanger - quatenaryaminoethyl (QAE)
This type of resin is positively charged
The resin binds negative proteins
Proteins are eluted by NaCl or altering pH - how does this work?
Cation exchanger
weak exchanger -
carboxymethy
(CM)
weak exchanger -
sulfipropyl (SP)
protein eluted by
the same means as
above
Size exclusion (SEC) or gel filtration chromatography
Media (solid phase) is a defined pore sizes in polymer beads,
large molecules “go around” small molecules “go through and around the
beads”
Smaller sized proteins are retained and come out last
Range of types of beads and chemistry - resin can be made of agarose,
acrylamide or other polymers
Size exclusion (SEC) or gel filtration chromatography
Can be used to separate proteins, remove salts exchange buffers or to
determine the molecular weight of a purified protein
2 Dimensional Electrophoresis
Combination of native or denatured PAGE and IEF
Run in two directions
1- PAGE - to separate by size
2- IEF to separate by charge alone
Good to separate very crude mixtures or determine the
difference between two proteins that are the same size
but with a different pI
2D-Electrophoresis
2D-electrophoresis allows separation of
proteins by both size and isoelectric
point. Each spot represents a different
protein. The horizontal represents the
isoelectric focusing direction, while the
veritcal represents the SDS PAGE
direction.
Immuno Analysis
Immunoglobins - 5 major classes main antibody response in
sera is IgG
antigen - foreign substance that triggers antibody formation
epitope - section of antigen that antibody recognizes
Antibodies consist of
heavy and light chains
Fab region - highly
variable - recognize
target (antigen)
FC heavy chain - interacts
with other proteins
polyclonal vs. monoclonal
antibodies
polyclonal
– from sera of an animal
– several epitopes to the same antigen
– some may cross react with other proteins in a non-
specific manner
– produce lots of antibodies al long as the animal lives and
you continue to boost
monoclonal
– derived from single cell - hybrid of mouse spleen and a
immortal cell line (lymphocyte and myeloma)
– inject mice then can grow cell in a dish
– antibodies purified from cell culture media
– single epitope, very specific
– unlimited production of antibodies
Antibodies in specific analysis
ELISA (Enzyme Linked ImmunoAssay)- most sensitive
detection methods for antibodies (aids test),
proteins, peptides and other substances (drug
testing)
Plastic Dish
Antibodies in specific analysis
ELISA (Enzyme Linked ImmunoAssay)- most sensitive
detection methods for antibodies (aids test),
proteins, peptides and other substances (drug
testing)
1 Protein of interest is
Plastic Dish Bound to plastic
Antibodies in specific analysis
ELISA (Enzyme Linked ImmunoAssay)- most sensitive
detection methods for antibodies (aids test),
proteins, peptides and other substances (drug
testing)
1 Protein of interest is
Plastic Dish Bound to plastic
Antibodies in specific analysis
ELISA (Enzyme Linked ImmunoAssay)- most sensitive
detection methods for antibodies (aids test),
proteins, peptides and other substances (drug
testing)
4
Secondary Antibody
Conjugated to an enzyme 2 Unreacted binding sites are
Covered with a non-reactive
protein
3 Primary Antibody
Recognizes Antigen 1 Protein of interest is
Plastic Dish Bound to plastic
Antibodies in specific analysis
ELISA (Enzyme Linked ImmunoAssay)- most sensitive
detection methods for antibodies (aids test),
proteins, peptides and other substances (drug
testing)
5
Enzyme reacts with
substrate producing
colored product
4
Secondary Antibody
Conjugated to an enzyme 2 Unreacted binding sites are
Covered with a non-reactive
protein
3 Primary Antibody
Recognizes Antigen 1 Protein of interest is
Plastic Dish Bound to plastic
Western blot - good for
mixtures of proteins,
identifying size and
characteristics
– transfer proteins form SDS
PAGE to paper for antibody
analysis.
– Primary antibody recognizes
protein antigen
– a secondary antibody
recognizes the Fc region and
is conjugated to a second
molecule to act as a signal
Characterization-1
Characterization-2
• Functional Studies
• Evidence of purity
Purpose:
– Determination of purity
– Determination of molecular mass
Characterization-4
Characterization-5