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Affinity Chromatography Affinity Chromatography

Affinity Chromatography - PowerPoint Presentation

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Uploaded On 2015-10-15

Affinity Chromatography - PPT Presentation

Supervisor Anton Zavialov By Elham Barazeghi Mehrafarin Ramezani Affinity Chromatography Separation of proteins by reversible interaction of proteins and specific ligand in matrix of column ID: 161520

affinity chromatography target elution chromatography affinity elution target proteins binding protein buffer matrix immobilized acid nucleic antibody column ligand

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Slide1

Affinity Chromatography

Supervisor:

Anton Zavialov

By:

Elham Barazeghi

Mehrafarin RamezaniSlide2

Affinity Chromatography:

Separation of proteins by reversible interaction of proteins and specific ligand in matrix of column

Advantages:SelectivityResolutionSuitable for concentrating a proteinSlide3

Some common terms :

Matrix

Spacer ArmLigandBindingElutionWash

Ligand couplingSlide4

Open and porous structure

Inert or non-specific interaction

OH group on sugar residues , good anchor for ligand attachmentAble to pass liquids through itself rapidlyStable enough in various conditionsSepharose is an ideal matrix!

MatrixSlide5

Improve efficiency of binding

length

Spacer armSlide6

How it works?

On the matrix:

on the chromatogram:

Slide7

Essential components of Affinity Chromatography system:

Column type(depending on target protein and the ligand)

Buffers:Loading bufferElution buffer Slide8

Elution methods:

pH elution

Ionic strength elutionCompetitive Polarity disturbanceDenaturing Slide9

Step vs. gradient elutionSlide10

Different types of Affinity Chromatography

Type of column

Target EnzymeSubstrate analogue, cofactor, inhibitorImmunoaffinityAntigen,

virus,cell

Lectin

Polysaccharides, glyco-proteins, cell surface receptor, cell

Nucleic acid

Complementary base sequence, histones, nucleic

acid polymerase, nucleic acid binding protein

Hormone, vitamin

Receptor, carrier protein

Glutathione

Glutathione-S

-

transferase

, GST fusion protein

Immobilized metal ion affinity chromatography (IMAC)

Poly (his)fusion proteins, native proteins with histidine, cystein or tryptophan residues on their surfacesSlide11

Immuno-affinity

chromatography (IAC)

A sub category of affinity chromatography A biologically related binding agent is used for the selective purification or analysis of a target compoundstationary phase consists of an antibody or antibody-related reagentThe selective and strong binding of antibodies for their given targets has made them of great interest

as

immobilized ligands in affinity

chromatographySlide12

Moser A.C., et. Al., Immunoaffinity chromatography: an introduction to applications and recent developments, bioanalysis -2010 ,2(4):769-790Slide13

The loading buffer has the ability to promote fast and efficient binding of the target analyte to immobilized antibodies which typically occurs under physiological conditions pH

=7

The elution carry out by temporarily lowering the effective strength of antibody binding to the target.Changing the mobile phase pH is the most popular method for eluting retained compounds from IAC columns.

This approach is usually conducted by applying an acidic buffer (pH 1–3) to the columnSlide14

An example of competitive

elution: http

://youtu.be/Hb791WsC78sSlide15

Thank You