affinity chromatography
TRANSCRIPT
AFFINITYAFFINITY CHROMATOGRAPHY CHROMATOGRAPHY
By: H. Nur HalipçiBy: H. Nur Halipçi
CHROMATOGRAPHYCHROMATOGRAPHY
ChromatographyChromatography is the is the collective term for a set ofcollective term for a set of lablaboratororatoryy techniques for the techniques for the separation of mixtures.separation of mixtures.
The term comes The term comes from Greefrom Greekk χρώμα:χρώμα:chromachroma means means color color and γραφειν:and γραφειν:grapheingraphein means means writewrite
INVENTED BYINVENTED BY
1903 – Tswett, a 1903 – Tswett, a Russian botanist Russian botanist coined the term coined the term chromatography. chromatography.
CHROMATOGRAPHYCHROMATOGRAPHY
ChromatographyChromatography is a physical is a physical method of separation in which the method of separation in which the components to be separated are components to be separated are distributed between two phases, one distributed between two phases, one of which is stationary (of which is stationary (immobilize immobilize phase) while the other (the mobile phase) while the other (the mobile phase) moves in a definite directionphase) moves in a definite direction
An animationAn animation
http://www.dnatube.com/video/2895/http://www.dnatube.com/video/2895/Column-ChromatographyColumn-Chromatography
TERMINOLOGYTERMINOLOGY
An An immobilized phaseimmobilized phase is a is a stationary stationary
phase which isphase which is immobilized on the support immobilized on the support
particles, or on the inner wall of the column particles, or on the inner wall of the column
tubingtubing
TERMINOLOGYTERMINOLOGY The The mobile phasemobile phase is the phase which is the phase which
moves in a definite direction. It may moves in a definite direction. It may be a liquid (LCbe a liquid (LC)), a gas (GC), or a , a gas (GC), or a supercritical fluid (SFC). A better supercritical fluid (SFC). A better definition:The mobile phase consists definition:The mobile phase consists of theof the samplesample beingbeing s separatedeparated and and the solvent that moves the sample the solvent that moves the sample through the column. through the column.
TERMINOLOGYTERMINOLOGY
TheThe analyteanalyte is the substance that is to is the substance that is to
bebe separatedseparated duringduring chromatography. chromatography.
TERMINOLOGYTERMINOLOGY
The The samplesample is the matter analysed in is the matter analysed in chromatography. It may consist of a chromatography. It may consist of a single component or it may be a single component or it may be a mixture of components. mixture of components.
TERMINOLOGYTERMINOLOGY
EElutionlution - washing of the mixture- washing of the mixture
EluentEluent - additional solvents used for elution- additional solvents used for elution
ResidencyResidency - time spent on column- time spent on column
Types of ChromatographyTypes of Chromatography
AFFINITY AFFINITY CHROMATOGRAPHYCHROMATOGRAPHY
Affinity HistoryAffinity History
1930s, first developed by A1930s, first developed by A..Wilhelm Wilhelm TiseliusTiselius-a swedish biochemist-a swedish biochemist, won the , won the Nobel Prize in 1948Nobel Prize in 1948
Used to study enzymes and other Used to study enzymes and other proteinsproteins
Relies on the affinity of various Relies on the affinity of various biochemical compounds with specific biochemical compounds with specific propertiesproperties
ExamplesExamples
• Antigen Antigen Antibody Antibody
• AntibodyAntibody AntigenAntigen
• SubstrateSubstrate EnzymeEnzyme
• DNA DNA HistonHiston
• HormoneHormone Binding Binding Protein/Receptor Protein/Receptor
Specificity of Affinity Specificity of Affinity ChromatographyChromatography
Specificity is based on three aspect of Specificity is based on three aspect of affinityaffinity
Matrix: for ligand attachment.
Spacer arm: used to bind ligand to matrix
Ligand: molecule that binds reversibly to a specific target molecule(site of interaction)
An animationAn animation
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So now what….So now what….
The Sample is injected into the The Sample is injected into the equilibrated affinity chromatography equilibrated affinity chromatography columncolumn
Only the substance with affinity for the Only the substance with affinity for the ligand are retained on the columnligand are retained on the column
The substance with no affinity to the The substance with no affinity to the ligand will elute offligand will elute off
The substances retained in the column can The substances retained in the column can be eluted off by changing the pH of salt or be eluted off by changing the pH of salt or organic solvent concentration of the eluentorganic solvent concentration of the eluent
MatrixMatrix
The matrix simply provides a structure to The matrix simply provides a structure to increase the surface area to which the molecule increase the surface area to which the molecule can bindcan bind
The matrix must be activated for the ligand to The matrix must be activated for the ligand to bind to it but still able to retain it’s own activation bind to it but still able to retain it’s own activation towards the target moleculetowards the target molecule
MatrixMatrix
Amino, hydroxyl, carbonyl and thio groups Amino, hydroxyl, carbonyl and thio groups located with the matrix serve as ligand binding located with the matrix serve as ligand binding sitessites
Matrix are made up of agarose and other Matrix are made up of agarose and other polysaccharidespolysaccharides
The matrix also must be able to withstand the The matrix also must be able to withstand the decontamination process of rinsing with sodium decontamination process of rinsing with sodium hydroxide or ureahydroxide or urea
LigandLigand The Ligand binds only to the desired molecule within the The Ligand binds only to the desired molecule within the
solutionsolution
The ligand attaches to the matrix which is made up of an The ligand attaches to the matrix which is made up of an inert substanceinert substance
The ligand should only interact with the desired molecule The ligand should only interact with the desired molecule and form a temporary bondand form a temporary bond
The ligand/molecule complex will remain in the column, The ligand/molecule complex will remain in the column, eluting everything else offeluting everything else off
The ligand/molecule complex dissociates by changing the pHThe ligand/molecule complex dissociates by changing the pH
Antibody affinityAntibody affinity(Immunoaffinity Chromatography)(Immunoaffinity Chromatography)
Used to purify antibody against a Used to purify antibody against a specific antigenspecific antigen
Ex:Immunoglobulins Ex:Immunoglobulins
PPurification of IgGurification of IgG, IgG fragments and , IgG fragments and subclasses subclasses havehave the high affinity of the high affinity of protein protein AA and and protein Gprotein G for the Fc region of for the Fc region of polyclonal and monoclonal IgG-type polyclonal and monoclonal IgG-type antibodiesantibodies
Protein A and protein GProtein A and protein G
Protein A and protein G are bacterial cell Protein A and protein G are bacterial cell surface proteins (from surface proteins (from Staphylococcus Staphylococcus aureus aureus and and StreptococcusStreptococcus respectively)respectively)
Recombinant protein A is available;Recombinant protein A is available; Engineered to include a C-terminalEngineered to include a C-terminal
Results in an enhanced binding capacityResults in an enhanced binding capacity
AFFINITY CHROMATOGRAPHY AFFINITY CHROMATOGRAPHY
Can be used;Can be used; Purify and concentrate a substance from a Purify and concentrate a substance from a
mixture into a buffering solution mixture into a buffering solution Reduce the amount of a substance in a Reduce the amount of a substance in a
mixture mixture Discern what biological compounds bind to Discern what biological compounds bind to
a particular substance, such as drugs a particular substance, such as drugs Purify and concentrate an enzyme solutionPurify and concentrate an enzyme solution
ApplicationsApplications
Used in Genetic Engineering Used in Genetic Engineering
- - nucleic acid purificationnucleic acid purification Production of VaccinesProduction of Vaccines
- - antibody purification from antibody purification from blood serumblood serum
And Basic Metabolic ResearchAnd Basic Metabolic Research
- - proteinprotein or enzyme purification or enzyme purification from cell free extractsfrom cell free extracts
Avidin(or strepdavidin) -biotin interaction is used Avidin(or strepdavidin) -biotin interaction is used to purify proteins to purify proteins
AvidinAvidin:protein deposited in the whites of :protein deposited in the whites of eggs(birds,reptiles…) eggs(birds,reptiles…)
StreptavidinStreptavidin is a tetrameric protein purified from is a tetrameric protein purified from the bacterium the bacterium Streptomyces avidiniiStreptomyces avidinii
Biotin:Biotin:((vitamin H or B7) cofactor in the metabolism vitamin H or B7) cofactor in the metabolism of fatty acids and leucine, and in gluconeogenesis of fatty acids and leucine, and in gluconeogenesis
PROTEIN PURIFICATIONPROTEIN PURIFICATION
The non-covalent bond The non-covalent bond formed between biotin and formed between biotin and avidin or streptavidin avidin or streptavidin
has a binding affinity >most has a binding affinity >most antigen and antibody bondsantigen and antibody bonds
~~strength of a covalent bond strength of a covalent bond
biotinylation biotinylation
means affinity chromatography using means affinity chromatography using immobilized avidin or streptavidin immobilized avidin or streptavidin
to separate the biotinylated protein to separate the biotinylated protein from a mixture of other proteins and from a mixture of other proteins and biochemicals biochemicals
Nucleic acid separation using immobilized Nucleic acid separation using immobilized
metal affinity chromatography (IMAC)metal affinity chromatography (IMAC)
The method can be used to purify compoundsThe method can be used to purify compounds
containing purine or pyrimidine moieties where containing purine or pyrimidine moieties where
the purine and pyrimidine moieties are shielded the purine and pyrimidine moieties are shielded
from interaction with the column matrix from from interaction with the column matrix from
compounds containing a non-shielded purine or compounds containing a non-shielded purine or
pyrimidine moiety or group. pyrimidine moiety or group.
Thus, double-stranded plasmid and genomic Thus, double-stranded plasmid and genomic DNA, which has no low binding affinity can be DNA, which has no low binding affinity can be easily separated from RNA or oligonucleotides easily separated from RNA or oligonucleotides which bind strongly to metal-charged which bind strongly to metal-charged chelating matrices chelating matrices
IMAC columns clarify plasmid DNA from IMAC columns clarify plasmid DNA from bacterial alkaline lysates, purify a bacterial alkaline lysates, purify a ribozyme, and remove primers and ribozyme, and remove primers and other contaminants from PCR reactions other contaminants from PCR reactions
ADVANTAGES OF AFFINITY ADVANTAGES OF AFFINITY CHROMATOGRAPHYCHROMATOGRAPHY
1) Extremely high specificity2) High degrees of purity can be
obtained 3) The process is very reproducible 4) The binding sites of biological
molecules can be simply investigated
DISADVANTAGES OF AFFINITY DISADVANTAGES OF AFFINITY CHROMATOGRAPHYCHROMATOGRAPHY
1) Expensive ligands2) Leakage of ligand3) Degradation of the solid support4) Limited lifetime5) Non-specific adsorption6) Relatively low productivity
REFERENCESREFERENCES
[1]http://en.wikipedia.org/wiki/[1]http://en.wikipedia.org/wiki/Affinity_chromatographyAffinity_chromatography
[2][2]www.apsu.edu/reedr/.../www.apsu.edu/reedr/.../AffinityAffinity%20%20ChromatographyChromatography%201.ppt %201.ppt
[3] [3] www.rpi.edu/dept/chem-eng/WWW/faculty/www.rpi.edu/dept/chem-eng/WWW/faculty/.../Lecture%2001.pdf -.../Lecture%2001.pdf -
[[4]4]www.chemistryinnovation.co.uk/.../www.chemistryinnovation.co.uk/.../Technology%20Area%20Technology%20Area%20AffinityAffinity%20%20ChromatographyChromatography.pdf -.pdf -
THANKS…THANKS…