ERT 313/4 BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

1. ELECTROPHORESIS ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011) ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

2. Electrophoresis refers to separation of compounds by employing electrophoretic mobility i.e. movement of charged molecules in response to an electric field. • Electrophoresis is carried out by adding the mixture of compounds to a conductive medium followed by the application of an electric field across the medium. • Positively charged molecules will migrate towards the negative electrode while the negatively charged compounds will migrate towards the positive electrode. • Neutral compounds will remain stationary. Proteins and nucleic acids have different charge on them depending on the medium pH. ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011) ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

3. In certain cases a compound can be imparted specific charge by using suitable chemicals e.g. ionic detergents. • Depending on the medium in which an electrophoretic separation is carried out electrophoresis can be classified into two types: • Gel electrophoresis • Liquid phase electrophoresis ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011) ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

4. Electrophoresis • A separation technique often applied to the analysis of biological or other polymeric samples • Among the most powerful for estimating purity because of its simplicity, speed, and high resolution, and also because there is only a small probability that any of the components being analyzed will be lost during the process of analysis • Has frequent application to analysis of proteins and DNA fragment mixtures and has been increasingly applied to the analysis of nonbiological and nonaqueous sample • The electric field doest not effect a molecule’s structure, and it is highly sensitive to small difference in molecular charge, size and sometimes shape ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

5. Electrophoresis Principles • The fundamental principle behind electrophoresis is the existence of charge separation between the surface of a particle and the fluid immediately surrounding it • An applied electric field acts on the resulting charge density, causing the particle to migrate and the fluid around the particle to flow • The electric fields exerts a force on the particle’s charge or surface potential • Two particles with different velocities will come to the rest in different locations after a fixed time in an electric field The particle velocity is related to the field strength by • There are two contributions to this apparent electrophoretic mobility: V: particle velocity, E: field strength or gradient (voltage per length), U: apparent electrophoretic mobility. (1) Uel: electrophoretic mobility of the charged particle Uo: the contribution from electroosmotic flow. (2) ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

6. Modes of Electropheretic Separation Gel electrophoresis • In gel electrophoresis, migration takes place though a gel slab • A common gel material for the study of proteins is cross-linked polyacrylamide • In most cases, the goal of experiment is to separate a sample according to molar masses of its components • However, the shape and charge will also determine the drift speed • One way to avoid this problem and to effect separation by molar mass is to denature the proteins in a controlled way • Sodium dodecyl sulfate is an anionic detergent that is very useful in this respect: it denatures proteins, whatever their initial shapes, into rods by forming a complex with them • Moreover, most proteins bind a constant amount of ion, so that the net charge per protein is well regulated • Under these conditions, different proteins in a mixture may be separated according to size only ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

7. Modes of Electropheretic Separation Figure 1 SDS-PAGE (denaturing gel electrophoresis) and Western blot results for bovine growth hormone (bGH) expressed as a C-terminal fusion to E. coli NusA protein. (a) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results with Coomassie blue staining. (b) Western blot results obtained by using rabbit anti-bGH polyclonal antibody and visualized by means of chemiluminescence. Fusion proteins were expressed at 37°C in E. coli by induction of the tac promoter. Equal portions of cell lysate, soluble fraction, and insoluble fraction were loaded. Key: m, markers; u, uninduced whole cell lysate; i, induced whole cell lysate; sol, soluble fraction; ib, inclusion body fraction. ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

8. Modes of Electropheretic Separation Capillary electrophoresis • The drift speeds attained by polymers in traditional electrophoresis methods are rather low; as a result, several hours are often necessary to effect good separation of complex mixtures • One way to increase the drift speed is to increase the electric field strength • However, there are limits to this strategy because very large electric fields can heat the large surfaces of an electrophoresis apparatus unevenly, leading to nonuniform distribution of electrophoretic mobilities and poor separation • In capillary electrophoresis, the sample is diepersed in a medium (such as methylcellulose) and held in a thin glass or plastic tube with diameters ranging from 20 to 100 µm • The small size of the apparatus makes it easy to dissipate heat when large electric fields are applied ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

9. Modes of Electropheretic Separation Figure 2 Separation of proteins by open tubular capillary electrophoresis, carried out in a 75 cm x 75 µm surface modified capillary at an applied voltage of 75 kV. Peak identities: A, egg white lysozyme; B, horse heart cytochrome c; C, bovine pancreatic ribonuclease a; D, bovine pancreatic α-chymotrypsinogen; F, equinemyoglobin. ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

10. Modes of Electropheretic Separation Isoelectric focusing • Naturally occurring macromolecules acquire a charge when dispersed in water • An important feature of proteins and other biopolymers is that their overall charge depends on the pH of the medium • For instance, in acidic environments protons attach to basic groups and the net charge is positive; in basic media the net charge is negative as a result of proton loss • At the isoelectric point, the pH is such that there is no net charge on the biopolymer • Consequently, the drift speed of the biopolymer depends on the pH of the medium, with s = 0 at the isoelectric point • Isoelectric focusing is an electrophoresis method that exploits the change of drift speed with pH • Consider a mixture of distinct proteins dispersed in a medium with a pH gradient along the direction of an applied electric field • Each protein in the mixture will stop moving at a position in the gradient where the pH is equal to the isoelectric point ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

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12. Support Media Paper Electrophoresis • One of the first matrices used for electrophoresis • In paper electrophoresis, the sample is applied directly to a zone on the dry paper, which is then moistened with a buffer solution before application of an electric field • Dyes are combined with samples and standards to help visualize the progress of the electrophoresis • The movement of samples on paper is best when the current flow is parallel to the fiber axis in the paper • Some advantages of paper are that it is readily available and easy to handle, requires no preparation, and allows the rapid development of new methodologies • Besides being easy to obtain, paper does not contain many of the bound charges that can interfere with the separation • A disadvantage of paper electrophoresis is that the porosity of commercial paper is not controlled, and therefore the technique is not very sensitive, nor is it easily reproducible ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

13. Support Media Polyacrylamide Gels • One of the most commonly used electrophoretic methods • Analytical uses of this technique center on protein nucleic acid characterization (e.g. purity, size, or molecular weight, and composition) • Acrylamide is neurotoxin, however, the reagents must be combined extremely carefully • The sieving properties of the gel are defined by the network of pores established during the polymerization : as the acrylamide concentration of the gel increases, the effective pore size decreases • The most commonly used combination of chemicals to produce a polyacrylamide gel is acrylamide, bisacrylamide, buffer, ammonium persulfate, and tetramethylenediamine (TEMED) ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

14. Support Media Agarose Gel • Agarose is a polymer extracted from red seaweed • When agar is extracted from the seaweed, it is in two components, agaropectin and agarose • The agarose portion is nearly uncharged, making it desirable for use as on electrophoresis matrix • The advantages of agarose electrophoresis are that it requires no additives or cross-linkers for polymerization, it is not hazardous, low concentration gels are relatively sturdy, and it is inexpensive • Commonly used for the separation of large molecules such as DNA fragments Capillaries • The fused silica capillaries are flexible due to an outer polyimide coating and are available in inner diameters ranging from 10 to 300 µm • Fused silica is transparent to UV light, which enables the capillary to serve as its own detection flow cell • Electrostatic interactions with the capillary surface can develop, however, when charged species are being separated • To overcome this problem is to chemically modify the inner capillary surface to produce a nonionic, hydrophilic coating, resulting in the shielding of the silanol functionalities ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

15. Support Media Comparison of Electrophoresis Matrices ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

16. Detection Techniques Chemical Staining • Incorporate a “fixing” step, such as a soak in dilute acetic acid for 1 h, etiher before or in conjunction with staining. • Frequently used stains: Coomassie brilliant blue (R250 and G250) and silver stain • The gel then is scanned with densitometer Fluorescence • Provides much better detection limit than simple chemical stains, typically involves the covalent binding of the fluorescent residue to the analyte • Fluorescamine- popular reagent for labeling of proteins • At room temp. and alkaline pH, fluorescamine can react with primary amine on the protein to generate a fluorescent derivative • The reagent ethidium bromide often used to visualize DNA ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)

17. Detection Techniques Radioactivity • If a sample is radioactive, the bands that separate during electrophoresis are subsequently radioactive • When the separation is complete, the electrophoretic matrix can be placed against x-ray film until the radiation makes a mirror image of the banding pattern on the film Immunoelectrophoretic Techniques • Known as “crossed immunoelectrophoresis” • A sample is first run longitudinally through an agarose gel for a predetermined time • Second, a longitudinal strip of the gel area in which of the sample was electrophoresed is typically cut out and placed into a similarly sized area of an antibody containing gel • As an electric current is applied to the gel, each band of the sample with form an antigen-antibody precipitation pattern through the gel ERT 313/4BIOSEPARATION ENGINEERING SEM 2 (2010/2011)