affinity chromatography

Download
Share  Add to  Flag Embed
Views:
 
Category: Education
     
 
License:   All Rights Reserved

Presentation Description

most useful

Comments

 

By: kmkodam (5 month(s) ago)

PLEASE ALLOW ME TO DOWNLOAD THE PRESENTATION ON AFFINITY CHROMATOGRAPHY

 

By: jilsy (7 month(s) ago)

good

 

By: vandanarohit (7 month(s) ago)

good presentation...

 

By: rathe (14 month(s) ago)

its nice and easy to understand

Presentation Transcript

Affinity Chromatography :

Affinity Chromatography Presented by: Parth Joshi* Paresh Desai,Hardik Patel, Chirag Baladhiya Department of pharmaceutics Atmiya Institute of pharmacy, Rajkot 1 Affinity chromatography

Contents::

Contents: Introduction Principle Materials Elution Detection Advantages Disadvantages Application Affinity chromatography 2

Introduction:

Introduction Affinity chromatography (AC) is a technique enabling purification of a biomolecule with respect to biological function or individual chemical structure. The substance to be purified is specifically and reversibly adsorbed to a ligand (binding substance), immobilized by a covalent bond to a chromatographic bed material (matrix). Purification by affinity chromatography is unlike all other techniques, In that it doesn't rely on differences in Biological property of molecule to be separated. 3 Affinity chromatography

Slide 4:

4 Affinity chromatography is a gel technique in which use is made of high specificity of biochemical reaction. A ligand which exhibits a specific affinity for particular compound gets covalently bonded to gel matrix and material is filled into the column. Choice of gel depends on type of group present in ligand molecule and nature of binding reaction with the substance to be purified. Samples are applied under favorable conditions for their specific binding to the ligand. Affinity chromatography

Slide 5:

5 Substances of interest are consequently bound to the ligand while unbound substances are washed away. Recovery of molecules of interest can be achieved by changing experimental conditions to favour desorption. Affinity chromatography media are commonly used for applications such as purification of fusion proteins, glycoproteins, nucleic acids ,immunoglobulins. Affinity chromatography

Principle:

Principle In this, The molecule of interest will have a well known and defined property which can be separated from complex mixtures in single process. The target molecule becoming trapped on a solid or stationary phase or medium. The other molecules in solution will not become trapped as they do not possess this property. The solid medium can then be removed from the mixture, washed and the target molecule released from the entrapment in a process known as elution . 6 Affinity chromatography

Slide 7:

Step-1 Binding of the selected ligand to the matrix requires that a covalent bond be formed between the two. As mentioned above, this is facilitated by derivitization of the sugar residues' hydroxyl groups. It is important to realize that the substrate might not be able to reach the ligand active site if it is hidden deep within the ligand. Therefore, most ligands are attached first to spacer arms which are then bonded to the matrix. The ligand-matrix gel is then loaded into an elution column. 7 Affinity chromatography

Slide 8:

8 Step 1: Attach ligand to column matrix Affinity chromatography

Slide 9:

Step-2 Once the column has been prepared, the mixture containing isolate is poured into the elution column. Gravity pulls the solution through the gel, because most of the proteins do not bind to the ligand-matrix complex. However, when the ligand's recognized substrate passes through the gel, it binds to the ligand-matrix complex, halting its passage through the gel. Some of the impurities flow through the gel due to gravity, but most remain, unbound, in the gel column. 9 Affinity chromatography

Slide 10:

10 Step 2: Load protein mixture onto column Affinity chromatography

Slide 11:

Step-3 In order to remove these unbound impurities, a wash of extreme pH, salt concentration, or temperature is run through the gel. It is important to use a strong wash so that all the impurities are removed. Once the impurities are washed-out, the only remaining part of the protein mixture should be the desired isolates. 11 Affinity chromatography

Slide 12:

12 Step 3: Proteins bind to ligands Affinity chromatography

Slide 13:

Step-4 Finally to collect isolate, which is still bound to the ligand-matrix in the gel, a stronger second wash is run through the column. This second wash relies on the reversible binding properties of the ligand, which allows the bound protein to dissociate from its ligand in the presence of this stronger wash. The protein is then free to run through the gel and be collected. 13 Affinity chromatography

Slide 14:

14 Step 4: Wash column to remove unwanted material, elute later Affinity chromatography

Slide 15:

15 Affinity chromatography

Materials :

Materials Major materials required for an affinity chromatography procedure are 1) a bead matrix 2) a ligand 3) a solution containing the substrate to be isolated 4) a wash to elute the non-bound impurities in the solution 5) a final wash to elute the bound substrate from its ligand. 16 Affinity chromatography

Slide 17:

17 Bead matrix Characteristics: It should exhibit good flow property. Mechanically & chemically stable and resistance against microorganisms can be attached easily by a variety of chemical reactions. Should posses functional groups to with ligands. Should be economical. Adequate partical size and shape e.g. increase in partical size reduces flow resistance and separation power Affinity chromatography

Examples of matrixs:

18 Examples of matrixs TYPE EXAMPLES Biopolymers Agarose Polysaccharides Cellulose,Starch Synthetic co-polymers Polyacrylamide, polystyrene Inorganic materials Porous glass,iron oxide Bio polymers/Synthetic co-polymers Agarose-polyacrylamide Inorganic materials/organic co-polymers Silica/Hydrophilic co-polymer Affinity chromatography

Slide 19:

Ligand The Ligand binds only to the desired molecule within the solution. The ligand attaches to the matrix which is made up of an inert substance. The ligand should only interact with the desired molecule and form a temporary bond. Affinity chromatography 19

Slide 20:

20 Ligand The ligand/molecule complex dissociates by changing the pH It is selected according to the desired isolate. For example, If isolate antibodies specific for antigen A from an antiserum, then antigen A as ligand, If isolate a specific enzyme then use either its substrate, an inhibitor, or even a cofactor. Next, one simply needs a mixture containing the desired isolate. Affinity chromatography

Characteristics of Ligand :

Characteristics of Ligand The ligand must be readily available. Ligand must be attachable (covalently) to the matrix (typically sepharose ) such that it still retains affinity for protein. Binding must not be too strong or weak. Elution involves passage of high salt or low pH buffer after binding. 21 Affinity chromatography

Slide 22:

Ligand Specificity AMP Enzymes with NAD cofactors an ATP dependent kinases Arginine Proteases such as prothrombin, kallikrein, clostripain Cibacron Blue Dye Serum Albumin, Preablumin Heparin Growth factors, cytokines, coagulation factors Protein A Fc region of immunoglobulins Calmodulin Calmodulin regulated kinases, cylcases and phosphatases EGTA-copper Proteins with poly-Histidine tails 22 Affinity chromatography

Slide 23:

23 Solution The solution is usually a protein rich mixture such as antiserum, which is poured into the elution column and allowed to run through the gel, at a controlled rate. Affinity chromatography

Prepacked columns :

Prepacked columns Prepacked columns: HiTrap Heparin HP (High performance) Column size: 5 × 1 mm 1 × 5 mm 5 × 5 mm Average particle diameter : 34μm Maximum operating flow rate: 4 ml/min 20 ml/min. Affinity chromatography 24

Slide 25:

25 Storage of pre-packed columns Store the pre-packed columns at 2-8 °C in an upright position with both caps in place. Thiomersal may be added for long term storage. DO NOT FREEZE Application areas : purification, isolation or removal of the following substances: Antithrombin III and other coagulation factors, lipoproteins, lipases, protein synthesis factors. Affinity chromatography

Elution:

Affinity chromatography 26 Elution Different method used for elution Nonspecific method Specific method Special method

Nonspecific method:

Affinity chromatography 27 Nonspecific method Change of pH, Ionic strength, temperature, polarity & by using diff. reagent like deforming eluents, urea, detergent. In many cases a change in pH leads to desorption especially complex binding proceed predominantly through ionic interaction. e.g. for proteases & inhibitors, adsorption occurs at 8.1 pH & elution at 3.1 pH

Specific method:

Affinity chromatography 28 Specific method Affinity elution (with substrate, cofactor, inhibitors ) Special method Electrophoresis, cleavage of matrix-ligand bond, buffer effect Electrophoresis is suited for desorption of antibiotics and hormone binding protein. By reductive cleavage of aza linkage with dithionate, Serum estradiol-binding protein are isolated from estradiol-agarose .

Slide 29:

Affinity chromatography 29 Detection of Antigen A method and device for detecting the presence of a suspected antigen in a sample whereby the antigen is trapped using affinity chromatography and the remaining eluant is differentially analyzed against a parallel processed control. The sample is first chromatographically segregated into distinct zones and the resulting eluant is split into two streams. One stream is contacted with a solid phase having immobilized capture ligands specific to the antigen of interest.

Slide 30:

Affinity chromatography 30 Detection of Antigen The other stream is passed through a similar solid phase where the capture ligands have been omitted. The two streams are then differentially analyzed, for example, with ultraviolet light absorption.

Slide 31:

Affinity chromatography 31 Detection of SNP Affinity chromatographic method for simple single nucleotide polymorphism (SNP) detection by use of a single-stranded DNA-coupled column and temperature gradient elution, utilizing the difference in thermal stability between hybridized double-stranded DNAs with and without mismatched base-pairs in the course of temperature gradient elution. The elution behavior of DNAs with and without SNPs in this chromatography and proposed a numerical calculation method based on a thermodynamic dissociation model.

Slide 32:

Affinity chromatography 32 Detection of SNP The effects of the column volume, flow rate of eluent and heating rate of the column on elution profiles were clarified. For designing DNA ligands, mismatched base-pair positions favorable for detection of SNPs were also explored by use of hybridized DNAs coding a part of the human TP53 gene.

Advantages:

Advantages High degrees of purity can be obtained. The process is very reproducible. The binding sites of biological molecules can be simply investigated. 33 Affinity chromatography

Disadvantages:

Disadvantages Cost, the difficulties associated with scale-up and the high labour intensity. With such drawbacks, affinity chromatography tends to be used in the purification of speciality chemicals and pharmaceutical compounds, where the need for high purity samples outweighs the cost of production. 34 Affinity chromatography

Application:

Affinity chromatography 35 Application The goal of affinity chromatography is to separate all the molecules of a particular specificity from the whole amount of molecules in a mixture such as a blood serum. For example, the antibodies in a serum sample specific for a particular antigenic determinant can be isolated by the use of affinity chromatography.

Step-1:

Affinity chromatography 37 Step-1 An immunoadsorbent is prepared. This consists of a solid matrix to which the antigen (shown in blue) has been coupled (usually covalently). Agarose, sephadex, derivatives of cellulose, or other polymers can be used as the matrix.

Step-2:

Affinity chromatography 38 Step-2 The serum is passed over the immunoadsorbent. As long as the capacity of the column is not exceeded, those antibodies in the mixture specific for the antigen (shown in red) will bind (noncovalently) and be retained. Antibodies of other specificities (green) and other serum proteins (yellow) will pass through unimpeded.

Step-3:

Affinity chromatography 39 Step-3 Elution. A reagent is passed into the column to release the antibodies from the immunoadsorbent. Buffers containing a high concentration of salts and/or low pH are often used to disrupt the noncovalent interactions between antibodies and antigen. A denaturing agent, such as 8 M urea, will also break the interaction by altering the configuration of the antigen-binding site of the antibody molecule. Another, approach is to elute with a soluble form of the antigen. These compete with the immunoadsorbent for the antigen-binding sites of the antibodies and release the antibodies to the fluid phase.

Step-4:

Affinity chromatography 40 Step-4 Dialysis: The eluate is then dialyzed against, for example, buffered saline in order to remove the reagent used for elution.

Slide 41:

Affinity chromatography 41

The procedure: :

The procedure: Synthesize a segment of DNA containing the sequence. Attach this artificial molecule to beads of an inert, solid medium (the matrix). Pour an extract of E. coli cells over the beads. Only molecules specific for the DNA sequence — in this case, molecules of the lac repressor — will bind to the beads. After irrelevant protein molecules have passed through the column, wash the beads with a buffer that will release the lac repressor molecules so they can be studied. Affinity chromatography 42

1.Separation of antibody::

1.Separation of antibody: Purification of anticobrotoxin antibody by affinity chromatography. Toxicon , Cobrotoxin was immobilized on Sepharose through its free amino groups without altering its antigenic activity. Modification of Arg-residues did not change the coupling capacity of cobrotoxin to Sepharose; however, the antigenic activity markedly decreased when Arg-30 and Arg-36 were modified

Slide 44:

Rabbits hyperimmunized with cobrotoxin in Freund's complete adjuvant produce non-precipitating as well as precipitating antibodies. By affinity chromatography of supernatants obtained from precipitin reaction at the maximum precipitation on a column of cobrotoxin-Sepharose, the non-precipitating antibodies were separated from the antisera. Both precipitating and non-precipitating antibodies were similar with regard to their molecular size and elution pattern on cobrotoxin-sepharose column. Specific neutralizing capacities of the non-precipitating antibody and its papain fragment increased 23- and 27·6-fold, respectively, over that of the antisera. These results may lead to a substantial improvement in the therapy of victims of snake bites.

2. Purification of Oligosaccharide Antigens by Weak Affinity Chromatography:

2. Purification of Oligosaccharide Antigens by Weak Affinity Chromatography Weak affinity (Ka≈102-104 M-1) monoclonal antibodies immobilized at high concentrations (> 50 mg/mL) on macroporous silica matrices provide a chromatographic medium for high performance separation of oligosaccharide antigens. Unlike classical affinity chromatography, weak affinity chromatography utilizes dynamic equilibrium kinetics as a basis for separation of antigens.

Slide 46:

Simple calculations based upon established principles of chromatographic theory enable rapid determination of affinity constants and systematic adjustment of column parameters to optimize analytical and semipreparative chromatography. Demonstrated applications of weak affinity chromatography include analysis of picomol quantities of oligosaccharides in complex biological fluids such as urine and serum and recovery of oligosaccharide antigens released from partially fractionated oligosaccharides of pooled human milk.

3. Aα and Aβ class II I-A determinants of antigen-specific T-helper factor and its antigen-nonbinding chain:

3. Aα and Aβ class II I-A determinants of antigen-specific T-helper factor and its antigen-nonbinding chain Antigen-specific T-helper factor (ThF) of CBA (H-2k) origin in the picryl (TNP) contact sensitivity system ( M r 60–70 kDa) was reduced with dithiothreitol under mild conditions. Affinity chromatography on antigen yielded an antigen-binding chain ( M r 20–30 kDa) and an antigen-nonbinding chain ( M r 40–50 kDa). Both chains were glycoproteins and were bound by lentil lectin. Affinity chromatography on anti-I-A monoclonal antibodies showed that I-A determinants occurred on the complete molecule and on the antigen-nonbinding, but not on the antigen-binding, chain.

Slide 48:

In contrast, five different monoclonal antibodies to I-Eα failed to absorb ThF. Moreover, the complete molecule and the I-A+ antigen-nonbinding chains had determinants of the alpha and beta chains of I-A and conformational determinants which are based on both chains. Sequential absorption and elution showed that Aα and Aβ determinants occurred on the same molecular complex. These data suggest a minimal model of ThF as a two-chain disulfide-bonded structure with an antigen-binding chain and a separate I-A+ antigen-nonbinding chain which behaves as a single unit in phosphate-buffered saline and has elements of both Aα and Aβ.

Slide 51:

IgG HC is susceptible to proteolytic cleavage in plant-based antibody production systems. Left: mAb6B5 murine IgG1 was recovered from tobacco leaves (pIgG) or cultured hybridoma cells (hIgG) by Protein G affinity chromatography and examined on nonreducing SDS-PAGE. Right: Bands corresponding to IgG, F(ab’)2, and Fab were then excised from the gels and reexamined on SDS-PAGE reducing gels to identify individual polypeptides. Lower: Antibody models correspond to the most abundant mAb species identified in tobacco. IgG, immunoglobulin G; HC, heavy chain; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; F(ab’)2, dimeric antigen binding fragment; Fab, antigen binding fragment; mAb, monoclonal antibodies; LC, light chain; CH, constant domain heavy chain; CH1, constant domain 1 of heavy chain; VH, variable domain of heavy chain; VL, variable domain of light chain; CL, constant domain of light chain.

Selected antibody forms relative to therapeutic applications. Each oval represents an immunoglobulin folding domain. Disulfide bonds are represented as red lines, and the polypeptide linker of the scFv is represented by a blue connecting ribbon. VL indicates variable domain light chain; VH, variable domain heavy chain; CL, constant domain light chain; CH, constant domain heavy chain; Fc, Fc fusion; IgG, immunoglobulin G; F(ab’)2, dimeric antigen binding fragment; Fab, antigen binding fragment; scFv, single chain antigen binding fragment.:

Selected antibody forms relative to therapeutic applications. Each oval represents an immunoglobulin folding domain. Disulfide bonds are represented as red lines, and the polypeptide linker of the scFv is represented by a blue connecting ribbon. VL indicates variable domain light chain; VH, variable domain heavy chain; CL, constant domain light chain; CH, constant domain heavy chain; Fc, Fc fusion; IgG, immunoglobulin G; F(ab’)2, dimeric antigen binding fragment; Fab, antigen binding fragment; scFv, single chain antigen binding fragment.

Slide 53:

Escherichia coli to express both scFv and Fab fragments. For Fab production, both antimethamphetamine and antiphencyclidine mAb were cloned into dual expression plasmids suitable for simultaneous expression of the light chain (LC) and corresponding heavy chain (HC) Fab domain (Fd). When a Strep affinity tag was placed at the C-terminus of the LC and a 6-histidine tag was placed at the C-terminus of the HC Fd, it was possible to select for fully assembled Fab using sequential affinity chromatography steps. Purified antimethamphetamine and antiphencyclidine Fab exhibit the correct size (~50 kDa), lack detectable bacterial contaminants, and possess the same affinity as the parent mAb.

Slide 54:

It should be noted that both the level of expression and the solubility of E coli –expressed scFv and Fab can vary greatly. Accumulation of soluble Fab required expression at 21°C. We found that higher growth temperatures limited the accumulation of soluble Fab and favored the formation of insoluble inclusion bodies. In addition, a similar antimethamphetamine scFv expressed in E coli formed insoluble inclusion bodies regardless of culture conditions examined and required protein refolding to acquire antigen binding properties.

Application :

Application Purification of substances from biological mixtures. Separation of native from denatured forms of proteins. Removal of small amounts of biomaterial from large amounts of contaminants. Purify and concentrate a molecule from a mixture in solution, even at very low concentrations. Reduce the amount of a substance in a mixture. Purify and concentrate an enzyme solution. 56 Affinity chromatography

Industrial Applications :

Industrial Applications Affinity chromatography is widely used in the pharmaceutical industry to purify and extract molecules of interest from complex mixtures. These molecules tend to be enzymes, proteins or amino acids, but other biological species can be selectively retained. Once isolated, these biological species can be selectively amplified to produce larger quantities, although at large concentrations. 57 Affinity chromatography

Slide 58:

Manufacturer of Antibody Sigma Aldrich Delta biolabs Icosagen AS Nooruse 9, 50411 Tartu, Estonia Call us at +372 737 7070 Span Diagnostics Limited ( India ) 173-B, New Industrial Estate, Udhna Mumbai - 394210, Maharashtra Dr. Reddy’s laboratory Chugai Pharmaceutical Bulk Antibody Production Plant, Utsunomiya, Japan Affinity chromatography 58

THANK YOU:

THANK YOU Affinity chromatography 59