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Chromatography for Protein purification 1. Ion-exchange chromatography 2. Hydrophobic chromatography 3. Affinity Chromatography 4. Size-exclusion / gel.

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Presentation on theme: "Chromatography for Protein purification 1. Ion-exchange chromatography 2. Hydrophobic chromatography 3. Affinity Chromatography 4. Size-exclusion / gel."— Presentation transcript:

1 Chromatography for Protein purification 1. Ion-exchange chromatography 2. Hydrophobic chromatography 3. Affinity Chromatography 4. Size-exclusion / gel permeation chromatography 1

2 Ion exchange chromatography  Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules. It is the most popular method for the purification of proteins and other charged molecules. It can be of two types: AEC (Anion exchange Chromatography in which negative charged molecules are attracted to a positively charged solid support and CEC (Cation exchange Chromatography in which the positively charged molecules are attracted to a negatively charged solid support). As the solid support ion-exchange resins are used and as the mobile phase buffer solution of varying P H are used. 2

3 Ion-exchange chromatography  The solution to be injected is usually called a sample, and the individually separated components are called analytes.  It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. It is often used in protein purification, water analysis. 3

4 Principle  Ion exchange chromatography retains analyte molecules based on ionic interactions. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge.  According to the type of ions exchanged it is subdivided into: 1. cation exchange chromatography 2. anion exchange chromatography 4

5 Ion Exchangers 5 Ion exchangers are usually cross-linked polymeric resins fixed with a charged functional group. Ion exchangers are usually cross-linked polymeric resins fixed with a charged functional group.

6 Ion exchangers – Functional groups Anion exchanger  Aminoethyl (AE - )  Diethylaminoethyl (DEAE - )  Quaternary aminoethyl (QAE - ) Cation exchanger  Carboxymethyl (CM - )  Phospho (PO - )  Sulphopropyl (SP - ) 6

7 Cation exchange chromatography  Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group. R-X C +M B R-X M + C + B _ _

8 Anion exchange chromatography  Anion exchange chromatography retains anions using positively charged functional group: R-X A +M B R-X B + M + A + + _ _

9 Procedure 1. A sample is introduced, either manually or with an autosampler, into a sample loop of known volume. 2. The mobile phase (buffered aqueous solution) carries the sample from the loop onto a column that contains some form of stationary phase material. 3. Stationary phase material is a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups. 9

10 Procedure 4. The target analytes (anions or cations) are retained on the stationary phase but can be eluted by increasing the concentration of a similarly charged species that will displace the analyte ions from the stationary phase. For example, in cation exchange chromatography, the positively charged analyte could be displaced by the addition of positively charged sodium ions. 10

11 MECHANISM OF ION-EXCHANGE (CATION EXCHANGE) CHROMATOGRAPHY OF AMINO ACIDS 11

12 Chromatography of Amino Acids 12

13 Procedure 5. The analytes of interest must then be detected by some means, typically by conductivity or UV/Visible light absorbance. 6. A chromatography data system (CDS) is usually needed to control an IEC. 13

14 Role of IEC in separating proteins  Proteins have numerous functional groups that can have both positive and negative charges.  Ion exchange chromatography separates proteins according to their net charge, which is dependent on the composition of the mobile phase. 14

15 Effect of pH in the separation of proteins  By adjusting the pH or the ionic concentration of the mobile phase, various protein molecules can be separated.  For example, if a protein has a net positive charge at pH 7, then it will bind to a column of negatively- charged beads, whereas a negatively charged protein would not. 15

16 Effect of pH in the separation of proteins  Proteins are charged molecules. At specific pH, it can exist in anionic (-), cationic (+) or zwitterion (no net charge) stage. cationic pH =pI anionic pH increase *pI isoelectric point 16

17 Choosing your ion-exchanger: know your proteins 1. Stability of proteins  stable below pI value, use cation-exchanger  stable above pI value, use anion-exchanger 2. Molecular size of proteins  <10,000 mw, use matrix of small pore size  10, ,000 mw, use Sepharose equivalent grade ( Sepharose is a trade name for a cross linked, beaded-form of a polysaccharide polymer material extracted from seaweed) 17

18  Important to consider the stability of proteins in choice of ion exchangers. Isoelectric focusing can be used to identify suitable ion-exchanger type 18 Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). It is a type of zone electrophoresis, usually performed on proteins in a gel, that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings. Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). It is a type of zone electrophoresis, usually performed on proteins in a gel, that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings.

19 Ion – exchange Chromatography

20 ELUTION  Done by washing the column with a strong salt solution (NaCl) which increases the ionic strength thereby pushing out the proteins.

21 HYDROPHOBIC CHROMATOGRAPHY  Principle Proteins are separated by hydrophobic interaction on columns with hydrophobic groups attached to the stationary phase material(e.g. phenyl-, octyl groups).  Surface hydrophobicity Hydrophobicity of amino acid side chains. Tryptophan > Isoleucine, Phenylalanine > Tyrosine > Leucine > Valine > Methionine Most hydrophobic side chains are buried in interior of protein, but some (clusters of) hydrophobic groups occur at surface of protein. Surface hydrophobic side chains can interact with hydrophobic groups for example attached to a column.

22 HYDROPHOBIC CHROMATOGRAPHY  Temperature Increasing temperature --> stronger hydrophobic interactions  Sample (application) Column having high concentration of a salt promotes binding (for example ammonium sulfate just below the concentration that starts to precipitate protein).  Elution of bound proteins Negative gradient of salting-out ions (from high to low concentration).

23 AFFINITY CHROMATOGRAPHY  In this type of chromatography, a compound with a special affinity (specialized compound) for the protein of interest is attached to the resin. For example, in immunoaffinity chromatography, antibodies to a specific protein (or its domain) are used as the specialized compound attached to the resin. The resin is then packed into a column. When a mixture of proteins is passed through the column, only those proteins with special affinity for the compound will stick to the column. All the other proteins will pass through the column. Once the non- specific proteins are eluted, proteins of interest that have stuck to the column can be eluted. These proteins can be removed by changing the ionic strength of the solution (so affecting the strength of binding of the protein to the column). Alternatively the special compound can be added to the elution solution and the equilibrium will change so that the protein will no longer stick to the column.

24 AFFINITY CHROMATOGRAPHY

25 SIZE EXCLUSION CHROMATOGRAPHY  It is also known as Gel Filtration. Used to separate proteins on the basis of their molecular weight. The column is packed with a porous resin.  The matrix retards proteins of different sizes for different periods. The proteins are collected automatically as they flow out of the column in tubes held in a fraction collector.  Larger proteins will be eluted first since the smaller proteins travel through the pores of the resin.

26 SIZE EXCLUSION CHROMATOGRAPHY


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