Download Miscellaneous Bioseparation

Miscellaneous
Bioseparation
Electrophoresis
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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
• Principles
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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
Electrophoresis
• The particle velocity is related to the field strength by
(1)
• where v is the particle velocity, E is the field strength or gradient (voltage per
length), and U is the apparent electrophoretic mobility.
• There are two contributions to this apparent electrophoretic mobility
(2)
• where Uel is the electrophoretic mobility of the charged particle and Uo is the
contribution from electroosmotic flow.
Modes of Electropheretic Separation
Gel electrophoresis
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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
The molar mass of each constituent protein is estimated by comparing its mobility in
its rod-like complexes form with a standard sample of known molar mass
However, molar masses obtained by this method are not as accurate as those
obtained by other techniques, such as MALDI-TOF and sixe exclusion
chromatography (SEC)
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.
Modes of Electropheretic Separation
Capillary electrophoresis
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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
Excellent separations may be effected in minutes rather than hours
Each polymer fraction emerging from the capillary can be characterized further by
other techniques, such as MALDI-TOF
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.
Modes of Electropheretic Separation
Isoelectric focusing
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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
In this manner, the protein mixture can be separated into its components
Support Media
Paper Electrophoresis
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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
Polyacrylamide Gels
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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)
Support Media
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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)
Agarose Gel
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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 crosslinkers 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
Support Media
Capillaries
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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
Comparison of Electrophoresis Matrices
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.
Some Elements of Cell Structure
Prokaryotic Cells
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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”
Figure 3 Diagrammatic representations of the structural features of the surfaces of
(a) Gram-positive and (b) Gram-negative bacteria. The membrane is also called the
plasma membrane or the cytoplasmic membrane.
Some Elements of Cell Structure
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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
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.
Some Elements of Cell Structure
Eukaryotic cells
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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
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,
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:
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(3)
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
• Enzymes and antibiotics – digestion of cell wall 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
Mechanical Methods for Cell Lysis
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Sonication
Ball milling
Pestle homogenization
Shearing devices (blender)
High pressure homogenizers
Bead mills
CeII Disintegration Techniques