Miscellaneous Bioseparation

1. Miscellaneous Bioseparation

2. 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

3. 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)

4. 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

5. 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.

6. 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

7. 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.

8. 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

10. 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

11. 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)

12. 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

13. Support Media Comparison of Electrophoresis Matrices

14. 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

15. 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

16. Cell Lysis • Cell disruption is a method or process for releasing biological molecules from inside a cell. • Cell lysis – breaking, or lysing, cells • Cell lysis is used mostly in western blotting to analyze the composition of specific proteins, lipids and nucleic acids individually or as complexes. Depending upon the detergent that is used either all membranes are lysed or certain membranes are lysed, leaving other membranes intact. For example if the cell membrane only is lysed then can be used to collect certain organelles - microscopy or western blotting.

17. Some Elements of Cell Structure Prokaryotic Cells • Cells that do not contain a membrane-enclosed nucleus are classified as either Eubacteria or Archaea which have its own potential • The bacteria cell envelope consists of an inner plasma membrane that separates all contents of the cell from the outside world, a peptidoglycan cell wall, and outer membrane • Bacteria cells with a very thick cell wall stain with crystal violet (Gram stain) and are called “Gram positive”, while those with thin cell wall stain very weakly – “Gram negative”

18. Some Elements of Cell Structure Figure 3 Diagrammatic representations of the structural features of the surfaces of Gram-positive and (b) Gram-negative bacteria. The membrane is also called the plasma membrane or the cytoplasmic membrane.

19. Some Elements of Cell Structure • Most biological membranes are phospholipids bilayers • The bacteria cell wall protects the plasma membrane and the cytoplasm from osmotic stress • The isoosmotic external concentration for most cells is 0.3 osmolar (osM) • Osmolarity refers to the molar concentration of all species in solution including ionized species • Higher concentrations outside the cell draw out water and cause the cytoplasm to shrink – “plasmolysis” • The cell wall is rigid, so the cytoplasm collapses within the plasma membrane while the wall does not • Concentration of salts and neutral solutes lower than 0.3 osM outside the cell force water into cell – “turgor” • Too much turgor can break the cell wall (lysis), a situation that can aid in cell disruption for retained bioproducts

20. Some Elements of Cell Structure Eukaryotic cells • Eukaryotic cells (cells with nuclei and internal organelles) are considerably more complicated than prokaryotic cells, and bioproducts may have to released from intracellular particles that are themselves coated with membranes and/or consist of large macromolecular aggregates • The eukraryotes includes fungi, and, of course, the higher plants and animals • The cell membrane of animal cells is easily broken, whereas the cell wall of plants is strong and relatively difficult to break

21. Some Elements of Cell Structure Figure 5 Eukaryotic cells. Simplified diagrammatic representation of an animal cell and a plant cell. The lysates of such cells contain the internal structures (organelles) shown,

22. Some Elements of Cell Structure Figure 4 (a) Phospholipid molecule and its outline. (b) Cell plasma membrane formed by phospholipids with their polar head groups in contact with aqueous phases. The molecules are about 2.5 nm long. Vertical bar represents a cholesterol molecule, and the large, irregular blob represents a protein molecule that is in contact with both the cytoplasm and the extracellular milieu.

23. Osmotic and Chemical Cell Lysis • Osmotic – drastic reduction in extracellular concentration of solutes • If the transmembrane osmotic pressure is due to solute concentration inside the cell and out, then the van’t Hoff law can be used to estimate this pressure, which apples to ideal, dilute solutions: • where π= osmotic transmembrane pressure, R = gas constant, T = absolute temperature (K), ci-co = difference between total solute molarity inside and outside the cell, respectively • The weakening or partial destruction of these walls can be achieved with chemical agents: detergents, chelators, enzymes, solvents and so on • Enzymes and antibiotics – digestion of cell wall to allow osmotic rupture or production of protoplasts • Detergents – break plasma membrane and are commonly used to lyse cultured animal cells • Nonionic detergents are used because they are far less denaturing proteins and other biological compounds than ionic detergents • Solvents – dissolve cell membrane and excess fat, may aid in precipitation (3)

24. Mechanical Methods for Cell Lysis • Sonication • Ball milling • Pestle homogenization • Shearing devices (blender) • High pressure homogenizers • Bead mills

25. Bead mill Cascading beads Rolling beads Cells being disrupted • Disruption takes place due to the grinding action of the rolling beads and the impact resulting from the cascading ones • Bead milling can generate substantial heat • Cryogenic bead milling : Liquid nitrogen or glycol cooled unit • Application: Yeast, animal and plant tissue • Small scale: Few kilograms of yeast cells per hour • Large scale: Hundreds of kilograms per hour.

26. Colloid mill Rotor Disrupted cells Stator Cell suspension • Typical rotation speeds: 10,000 to 50,000 rpm • Mechanism of cell disruption: High shear and turbulence • Application: Tissue based material • Single or multi-pass operation

27. French press Plunger Cell suspension Cylinder Jet Orifice Impact plate • Application: Small-scale recovery of intracellular proteins and DNA from bacterial and plant cells • Primary mechanism: High shear rates within the orifice • Secondary mechanism: Impingement • Operating pressure: 10,000 to 50,000 psig

28. Ultrasonic vibrations Ultrasound generator Ultrasound tip Cell suspension • Application: Bacterial and fungal cells • Mechanism: Cavitation followed by shock waves • Frequency: 20 kHz • Time: Bacterial cells 30 to 60 seconds, yeast cells 2 to 10 mins. • Used in conjunction with chemical methods

29. CeII Disintegration Techniques