Seperation Techniques for Biomolecules Humaira Rafique 2011-ag-1158

Contents: Chromatography Electrophoresis Microfilteration

Chromatography

Chromatography Technique to separate components of a mixture by passing them through a matrix. Separation occurs because each compound in a mixture interacts differently with the matrix.

Types of Chromatography: Column Chromatography Size Exclusion Chromatography Affinity chromatography Ion Exchange chromatography High Performance Liquid Chromatography Fast Performance Liquid Chromatography Gas Chromatography

Column chromatography Compounds are separated through differential intermolecular forces between the components of the mixture with the mobile phase and between the components with the stationary phase.

COLUMN CHROMATOGRAPHY Polar components (b) adsorb more strongly to the polar silica and elute after the less polar components (a), which move more quickly with the non-polar (relative to silica) solvent.

Column chromatography Applications: Column chromatography (CC) is an extremely valuable technique for purification of Synthetic or Natural products.

Size Exclusion Chromatography Size Exclusion chromatography is a separation based on size. It is also called molecular exclusion, Gel Filteration or gel permeation chromatography.

Size Exclusion Chromatography Separation mechanism is not based on adsorption. Resolution depends on particle size, pore size, flow rate, column length and diameter, and sample volume. Three steps Equilibration Sample loading Elution

Size Exclusion Chromatography Applications: Fractionation and molecular weight determination of proteins, nucleic acid separation, plasmid purification, polysaccharide fractionation

Affintiy Chromatography Affinity chromatography (AC) is a technique enabling purification of a biomolecules 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).

Affinity Chromatography Two important things to consider; Finding a ligand specific enough to allow step elution. Finding conditions for safe binding and release within the stability window of the target molecule and the ligand.

Affinity Chromatography Applications: It is used to separate and purify the Proteins Peptides DNA fragments, etc

Ion Exchange chromatography Separates compounds based on their net charges. Negatively or positively charged functional groups are covalently bound to a solid support, yielding either a cation or anion exchanger, respectively.

Ion Exchange chromatography Applications: It is used to separate the 4 Heamoglobin variants Proteins, etc

High Performance Liquid Chromatography (HPLC) HPLC is also called high-pressure liquid chromatography.  It is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying and purifying the individual components of the mixture.

High Performance Liquid Chromatography

High Performance Liquid Chromatography Applications: HPLC has many uses including Medical .Detecting vitamin D Concentrations in blood serum Legal Detecting performance enhancement drugs in urine

High Performance Liquid Chromatography Applications: Research Purifying substances from a complex biological sample Separating similar synthetic chemicals from each other Manufacturing (e.g. during the production process of pharmaceutical and biologic products

Fast Performance Liquid Chromatography (FPLC) Liquid chromatography is a term which refers to all chromatographic methods in which the mobile phase is liquid. The stationary phase may be a liquid or a solid, in the form of a matrix A type of liquid chromatography where the solvent velocity is controlled by pumps. The pumps control the constant flow rate of the solvents

Fast Performance Liquid Chromatography Applications: It is used for Purification of proteins Rapid purification of RNA Analyzing lipoprotein profiles In pharmaceutical (eg, Drugs) The analysis of chemical components of Chinese herbal medicines

Gas Chromatography The mobile phase of a chromatographic system is gaseous and stationary phase is a liquid coated on inert solid particles, the technique is called Gas Liquid Chromatography or simple Gas chromatography.

Gas Chromatography Applications: Analyze the contents of chemical products Volatile mixtures are separated Quality assessment of alcoholic beverages Analysis of food Analysis of biomolecules(eg, melanins)

Other Types of Chromatography Hydroxyapatite chromatography Reverse-Flow Chromatography Hydrophobic interaction chromatography Immobilized metal ion affinity chromatography

Hydroxyapatite chromatography Ca5(PO4)3(OH)2 is a form of calcium phosphate that can be used for the separation and purification of proteins enzymes nucleic acids viruses, and other macromolecules.

Reverse-Flow Chromatography Flow to the column is then reversed and the bound proteins elute from the top of the column in very concentrated form, which helps prevent denaturation. Especially useful when purifying antibodies using Protein A or other affinity columns.

Hydrophobic interaction chromatography Separates molecules based on their hydrophobicity. Sample molecules containing hydrophobic and hydrophilic regions are applied to an HIC column in a high-salt buffer. Usually a decreasing salt gradient is used to elute samples from the column in order of increasing hydrophobicity.

Immobilized metal ion affinity chromatography Most widely used – nickel: also zinc and cobalt Nickel binds molecules rich in electrons – such as histidine (from the His-tag fame!) IMAC uses the affinity of histidine’s imidazole side chains for metal ions.

Electrophoresis

Electrophoresis s Electrophoresis is a separation method based on the differential rate of migration of charged species in a buffer solution across which has been applied a dc electric field. The rate of migration of a given species depends upon charge and size of ions. dc electric is a separation method based on differential rate of migration of charged species in a buffer solution across which has been applied a dc electric field.

Types of Electrophoresis Gel Electrophoresis SDS-PAGE Electrophoresis Immuno Electrophoresis Capillary Electrophoresis

Gel Electrophoresis Gel electrophoresis separates molecules on the basis of their charge and size.The charged macromolecules migrate across a span of gel because they are placed in an electrical field. The gel acts as a sieve to retard the passage of molecules according to their size and shape.

How does gel electrophoresis work? The gel is made from agar DNA is a negative molecules Molecules sort based on Charge Size shape

The movement of molecules is impeded in the gel so that molecules will collect or form a band according to their speed of migration. % agarose: 2% 4% 5% 500 bp 200 bp 50 bp 500 bp 200 bp 50 bp 500 bp 200 bp 50 bp The concentration of gel/buffer will affect the resolution of fragments of different size ranges.

Gel Electrophoresis Types: Slab Gel Electrophoresis (SGE) Polyacrylamide Gel Electrophoresis (PAGE) Agarose Gel Electrophoresis (AGE) Pulsed Field Gel Electrophoresis (PFGE)

Slab Gel Electrophoresis Slab separation are carried out on a thin flat layer or slab of a porous semisolid gel containing an aqueous buffer solution within its pores

Slab Gel Electrophoresis Slab gel electrophoresis can have either a horizontal or vertical format. Sample is introduced into wells at the top of the gel.

Slab Electrophoresis Limitations: Slow Labor intensive Difficult to automate Does not yield very precise Quantitative information Joule heating

Polyacrylamide Gel Electrophoresis Acrylamide, in combination with a cross linker, methylene bis-acrylamide Synthetic, consistent polymer Polymerization catalysts: ammonium persulfate (APS) plus N,N,N',N'-tetramethylethylenediamine (TEMED), or light activation

PAGE Resolves 1 bp difference in a 1 kb molecule (0.1% difference), Tracking and loading dyes are used. Different buffers are used for protein separation. Tris Borate EDTA (TBE), Tris Acetate EDTA (TAE), Tris Phosphate EDTA (TPE) used most often for DNA.

Native PAGE (non-denaturing PAGE) Types of PAGE Native PAGE (non-denaturing PAGE) The proteins are resolved in the absence of SDS SDS-PAGE (Denaturing PAGE) The proteins are resolved in the presence of SDS

Feature of PAGE High resolving power. Acceptance relatively large sample size. Minimal interaction b/w migrating molecules & matrix. Physical stability of matrix. Separation basis Molecular sieving Electrophoresis mobility

Agarose Gel Electrophoresis Agarose gel electrophoresis is routinely used for the preparation and analysis of DNA. It is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. We will be using agarose gel electrophoresis to determine the presence and size of PCR products.

Agarose Gel Electrophoresis

SDS-PAGE What is SDS-PAGE? Based on the migration of charged molecules in an electric field Separation technique Uses the Polyacrylamide gel as a “support matrix”. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run.

Role of SDS Denatures proteins by wrapping around the polypeptide backbone. SDS binds to most proteins in amount roughly proportional to molecular weight of the protein-about one molecule of SDS for every two amino acids In doing so, SDS creates a large negative charge to the polypeptide in proportion to its length

Conclusion SDS PAGE is a useful method for separating and characterising proteins, where a researcher can quickly check the purity of a particular protein or work out the different number of proteins in a mixture. Since we did not obtain results for the experiment, we have to rely on sample results Cannot validate the experimental technique

Pulsed Field Gel Electrophoresis Very Large DNA Molecules are Separated by Pulsed Field Gel Electrophoresis (PFGE).

Immuno Electrophoresis/ID Ag-Ab reaction Affinity based Agarose 1 %

Capillary Electrophoresis Capillary electrophoresis yields high-speed, high-resolution separation on exceptionally small sample volumes

Modes of Capillary Electrophoresis Capillary Zone Electrophoresis Capillary Gel Electrophoresis Capillary Isotachophoresis Capillary Isoelectric Focusing

Capillary Electrophoresis

Electrophoresis Applications: It has been applied to a variety of difficult analytical separation problems Inorganic anions and cations Amino acids Vitamins Drugs

Electrophoresis Applications: Carbohydrates Peptides Nucleotides Proteins Polynucleotides

Summary Electrophoresis is used to separate molecules by size and/or charge. Nucleic acid fragments can be resolved on agarose of polyacrylamide gels. PFGE is used to resolve very large DNA fragments. CGE is more rapid and automated than slab gel electrophoresis. The choice of electrophoresis method depends on the type and size of sample.

Microfilteration

Microfiltration Separates soluble contaminants remaining within the supernatant Supernatant may include: Other proteins Bio-molecules Un-used growth media Microfiltration image from: http://www.aaflow.de/filtertech/index.html

Microfiltration MF membranes are easily tested by direct examination, as their pores can be observed by electron microscopy. Large areas of microfiltration membrane can be tested and verified by a bubble test: Pores of the membrane are filled with liquid, then a gas is forced against the face of the membrane.

How does Microfiltration work? Pressure driven process Separates: Components in a solution or suspension based on molecular size Particles size range: 10mm (starches) to aprx. 0.04mm (DNA, Viruses, and globular proteins) Microfiltration image from: http://www.faireymicrofiltrex.com/Vokes%20Microfiltration/media/images/e-fluor.gif

Microfiltration

Microfiltration Proteins act as the permeate Applications: Proteins act as the permeate Separates larger particles For example- Colloids Fat globules Cells

Microfiltration Applications: Pharmaceutical, Biotechnology, Medical app’s (used on the vents of sterile water tanks to prevent microbial contamination; protect autoclaves and freeze-dryers during the admission of gas after the duty cycle; etc.)

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