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Buffers and Buffering
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Theory
How to Prepare a Buffer
Common Buffers
Common Buffer Preparations
Buffer Theory
Most biological systems will function only within a quite narrow range of conditions, and
their activity can vary widely within that range. The acidity, or free proton concentration,
of the environment is an important parameter. To prevent the proton concentration of a
solution from changing, compounds can be added to a solution that "buffer" or minimize
such changes. A compound will act as a proton concentration buffer if it limits changes
in proton concentration by binding protons when the proton concentration of the solution
increases and releasing bound protons when the proton concentration decreases (Eqn 1).
+
H+ + Buffer
(Eqn 1)
Unfortunately, any one compound will be effective as a buffer only for a limited range of
proton concentrations, and so the first step in preparing a buffer is deciding which buffer
+
to use.
)
and half in the base form (Buffer), i.e., when half of its proton binding sites are filled, a
buffer should be chosen that will be about half filled at the proton concentration desired.
The affinity of a compounds for protons is often expressed as its acid dissociation
constant (Ka), defined in Eqn 2. This is convenient because, as can be seen from Eqn 2,
the value of the acid dissociation constant is equivalent to the proton concentration of a
solution at which the compound will have half of its proton binding sites filled,
(Eqn 2)
If the proton concentration and the acid dissociation constant are both expressed as their
negative log, Eqn 2 becomes;
(Eqn 3)
and if, for reasons largely historical, the negative log operator is called "p", the
expression becomes;
(Eqn 4)
rearranging Eqn 4 and inserting the values for the pH and pKa;
(Eqn 5)
Further rearranging of Eqn 5 gives Eqn 6;
(Eqn 6)
After choosing a buffer, the next step is to decide its concentration. The buffer
concentration must be sufficient to maintain the pH within acceptable limits with the
changes in proton concentration expected to occur. For biological systems, this generally
means that the total buffer concentration ([Buffer]total) is within a range from 1 mM to
200 mM. Knowing that the total buffer concentration is equal to the sum of the
concentrations of its forms;
(Eqn 7)
by substituting for the [Buffer] term in Eqn 7 with its equivalent from Eqn 6, we see;
(Eqn 8)
and by rearranging,
(Eqn 9)
So for any pH we choose, by finding a buffer whose pKa is within around one pH unit of
that pH,
Common buffers
Molecular Weights of Different Forms
Buffer
pKa
Neutral
form
HCl salt
Na+ salt
H3PO4 / NaH2PO4 (pKa1)
2.12
98.0
-
120.0
Glycine (pKa1)
2.34
75.07
111.5
-
Citric acid (pKa1)
3.13
Acetic acid
4.75
60.05
-
82.0
Citric acid (pKa2)
4.76
192.1
-
294.1
MES
6.15
195.2
-
217.2
Cacodylic acid
6.27
H2CO3 / NaHCO3 (pKa1)
6.37
62.01
-
84.01
Citric acid (pKa3)
6.40
Bis-Tris
6.50
209.2
245.7
-
ADA
6.60
190.2
-
212.1
Bis-Tris Propane (pKa1)
6.80
282.4
318.9
-
PIPES
6.80
302.4
-
325.3
ACES
6.90
182.2
218.7
-
Imidazole
7.00
68.1
104.5
-
BES
7.15
213.2
-
235.2
MOPS
7.20
209.3
-
231.2
NaH2PO4 / Na2HPO4 (pKa2)
7.21
120.0*
-
142.0*
TES
7.50
229.3
-
251.2
HEPES
7.55
238.3
-
260.3
HEPPSO
7.80
268.3
-
290.3
Triethanolamine
7.80
149.2
185.7
-
Tricine
8.10
179.2
215.6
-
Tris
8.10
121.1
157.6
-
Glycine amide
8.20
74.1
110.6
-
Bicine
8.35
163.2
199.7
-
Glycylglycine (pKa2)
8.40
132.1
168.6
-
TAPS
8.40
243.2
-
265.3
Bis-Tris Propane (pKa2)
9.00
?
355.3
-
Boric acid (H3BO3 / Na2B4O7)
9.24
61.8
-
201.24
CHES
9.50
207.3
-
229.3
Glycine (pKa2)
9.60
75.07
-
96.1
NaHCO3 / Na2CO3 (pKa2)
10.25
84.01
-
105.99
CAPS
10.40
221.3
-
243.3
Piperidine
11.12
Na2HPO4 / Na3PO4 (pKa3)
12.67
142.0*
-
164.0*
*The anhydrous molecular weight is reported in the table. Actual molecular weight will
depend on the degree of hydration.
PROTEIN PURIFICATION AND ANALYTICAL TECHNIQUES
-Studies on pure proteins are essential for understanding structural and functional
properties of proteins.
-Method for each protein worked out by trial and error on small samples

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goal: separate the protein you want from other proteins and small molecules
mild conditions to avoid denaturation (usually low temperature, 0–4° C, and
avoiding extremes of pH)
need detection method (e.g. biological activity, or spectroscopy)
usually use several purification methods, one after another
start with (mixture of) proteins in buffered solution, e.g. extract of proteins
from cells that have been lysed (broken open)
Source of Protein
In order to purify a protein you need a source: could be blood or some other biological
fluid, but most often whole cells, usually a specific type (liver, muscle, yeast, bacteria,
etc.)


Cells must be broken open (lysed, e.g., by osmotic shock or by mechanical
disruption such as with a "French press" or a tissue homogenizer) to disrupt cell
membranes to release proteins in soluble form without damaging the protein.
Membrane-bound proteins can also be purified, but different approaches are
required.
Initial fractionation of homogenate


usually by differential centrifugation --> several fractions (successive pellets,
supernatants) of decreasing density, each with lots of proteins
assay each fraction to find which fraction contains most of the protein of interest,
and fractionate that further by more discriminating methods.
CHROMATOGRAPHIC TECHNIQUES

Column Chromatography
o Invention of column chromatography a critical event in biochemistry,
because it was the basis for development of procedures for obtaining pure
proteins.
o Different kinds of chromatographic separations based on one of the
following:
 size of protein (molecular sieve chromatography = gel filtration =
size exclusion chromatography), or
 net charge of protein (ion exchange chromatography), or
 specific ligand binding properties of protein (affinity
chromatography)
o
o
o
o
o
In column chromatography a solid phase ("matrix", "resin", generally
some kind of polymer, often a polysaccharide)(see below) is placed in a
glass tube, the column.
terminology:
 adsorbent: solid material/matrix, a "stationary phase" that some
molecules bind to (adsorb to)
 elution: the process of washing something off an adsorbent (with
an eluting buffer; the solution coming off the column is the
eluate.)
Protein mixture is
passed into the
column.
Either due to
molecular size
differences or
different binding
affinities for
column matrix,
some proteins are
retained longer on
the column (e.g.,
some bind more
tightly than
others)
The properties of some different types of column packing materials (for separations
based on molecular size, charge or specific ligand binding) are described below.

Gel Filtration Chromatography (also called "Molecular Sieve"
chromatography, or "Size Exclusion" chromatography)
o stationary phase (column matrix) = "beads" of a polysaccharide material
that separates proteins based on size and shape.
 Different column packing materials (hydrated, porous beads of
carbohydrate polymer (e.g. dextran or agarose) or polyacrylamide)
available, with wide range of molecular exclusion limits, for
separating proteins of all sizes.
 Solution of mixture of proteins, small molecules, etc. "filters"
through the beads:
 Large molecules can’t get into the smaller pores in the
beads and move more rapidly through the column,
emerging (eluting) sooner.
 Smaller molecules and ions can enter all the pores in the
beads with the buffer, and thus have more space to
"explore" on their way down the column, and elute later.
o
For any particular column dimensions and material, volume of buffer
required to elute a specific protein depends mostly on molecular weight
of the protein (but shape plays an important role also -- separation is
really based on differences in hydrodynamic volume). Thus, one can
separate proteins by size.
o
Size exclusion chromatography
o
This animation illustrates how size
exclusion chromatography works.
Note how the small red spheres p
into the channels in the beads,
whereas the large blue spheres do
not. Thus, the small spheres have
longer "distance" to transverse than
large spheres to get to bottom of
column, which means that a larger
volume of solvent must pass throu
the column before the red spheres
eluted.
o
The following plot of relative amo
of the large solute (blue) and of th
smaller solute (red) goes with the
animation.
Larger solutes elute EARLIER,
smaller solutes LATER, from a si
exclusion column.
o
o
o
calibrate the column:

determine elution volumes of proteins with
known molecular weights
 
construct a calibration curve relating(known)
molecular weight to (measured) elution volume
specifically for that column.

o

o
Such a calibration curve can then be used to estimate
the molecular weight of an unknown protein.
Ion Exchange Chromatography
o
o
o
o
o
o
o
Ion exchange resins have charged groups covalently attached to the
stationary phase (adsorbent, matrix), either positive or negative.
Obviously, if ionizable groups are weak acids or bases, the pH of the
buffer determines the charge state of the matrix.
Proteins bind to the matrix by electrostatic interactions.
Strength of these interactions depends on
 net charge on the protein (a function of buffer pH and the nature
of the ionizable groups on that protein, reflected in the pI of the
protein), and
 salt concentration of the buffer (high salt concentrations reduce
the interaction and can be used to elute the proteins by competing
with the protein groups for binding to the charged groups on the
matrix).
The higher the net charge on the protein at the pH of the environment on
the column, the more tightly it sticks to an oppositely charged matrix,
and the higher the salt concentration required to elute it from the
column.
The further the "working pH" is from the isoelectric point (pI) of a
protein, the greater the net charge on the protein, and the more
tightly it will stick to an ion exchanger of opposite charge.
By proper choice of eluting buffer (often a gradient with increasing salt
concentation, or changing the pH), specific proteins can be eluted from
the column and separated from other proteins in the mixture.
Ion Exchange Chromatography
 Example in figure is cation exchange chromatography -- column
packing beads have covalently attached negatively charged groups
 Negatively charged solutes move down the column more or less
without sticking, so they elute first.
 Positively charged solutes bind, and the higher the positive charge
on a molecule, the tighter it binds, so the later it elutes.


Example: Suppose you have 5 different proteins, with
relative isoelectric points as indicated on the pH scale below.
pH SCALE (working pH = 6.5 for these examples):
0 -----------pI#5----------pI#4------- 6.5 --------pI#1----------pI#2----------pI#3------------- 14
Suppose that your column is equilibrated and being
eluted at pH 6.5 (the working pH is 6.5), by washing
the column with a gradient of buffer of increasing salt
concentration.
Protein
What's the RELATIVE net charge at pH
6.5?
1
2
3
4
5
o
o

ANION EXCHANGE
Anion exchange matrix has + charged groups (e.g., DEAE
(diethylaminoethyl) groups).
 A molecule with a net + charge won't stick, so will wash on
through and elute before anything else (proteins 1, 2 and 3
in the current example).
 Molecules with net - charge will elute in the order of their
pI values, because of differences in net charge: the most charged one (the one whose pI is furthest from the working
pH) sticks the most tightly (elutes last). See elution profile
below.
CATION EXCHANGE
Cation exchange matrix has – charged groups (e.g.,
carboxymethyl (CM) groups).
 
A molecule with a net - charge won't stick, so
will wash on through and elute before anything else
(proteins 4 and 5 in the current example).
 
Molecules with net + charge will elute in the
order of their pI values, because of differences in
net charge: the most + charged one (the one whose
pI is furthest from the working pH) sticks the most
tightly (elutes last). See elution profile below.
Label the peaks below with #1, #2, #3, #4, and/or #5,
based on the expected order of elution of proteins #1-5
from a cation exchange column, or from an anion
exchange column, at pH 6.5.

Affinity Chromatography
o a more specific adsorbent in which a ligand specifically recognized by
the protein of interest is covalently attached to the column material
o When a mixture of proteins is passed through the column, only those few
that bind strongly to the ligand stick, while the others pass through the
column.
o Protein of interest is eluted with a buffer containing the free ligand,
which competes with the column ligand to bind to the protein, and protein
washes off (with bound ligand).
o Affinity Chromatography
o
some variations:


immunoaffinity chromatography: an antibody specific for a protein
is immobilized on the column and used to affinity purify the
specific protein.
"polyHis tags" on recombinant proteins: a sequence of His residues
is placed (by genetic engineering of a cloned gene) at the Cterminus of a specific recombinant protein to be produced in vivo,
and that protein can be purified on a column with Ni2+ ions (or
Cu2+ or Co2+ or Zn2+) held in chelated form on an affinity column;
the His imidazole groups on the end of the recombinant protein
bind with high affinity, but other proteins don't stick. The
recombinant protein can then be eluted with an imidazole buffer.
ELECTROPHORESIS
Electrophoresis
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

In an electric field, a protein or
other charged macromolecule will
move with a velocity that depends
directly on the charge on the
macromolecule and inversely on
its size and shape.
pH obviously important in
determining net charge
Gel electrophoresis is carried out
in some supporting media, usually
polyacrylamide or agarose, with
pores of big enough to allow
passage of the macromolecule.
Electric field is applied, and
molecules move toward electrode
opposite to their net charge, but
they’re slowed down ("friction")
by the gel
o larger or more elongated
shaped molecules move the
most slowly
o smaller, most compact
molecules move faster.
The proteins in the gel are easily
stained for detection purposes.
Because the net charge on a
protein and its molecular weight
are characteristic properties of a
protein, electrophoresis is a
powerful method for
characterizing degree of purity of
a protein preparation, but can also
be used for purification of small
amounts of proteins.

Discontinuous Gel Electrophoresis ("disc gel electrophoresis")
3 experimental variations to ordinary gel electrophoresis:
1) 2 gel layers, a lower or resolving gel and an upper or stacking gel
2) The buffers used to prepare the 2 gel layers are of different ionic strengths and
pH
3) The stacking gel (upper gel) has a lower acrylamide concentration, so its pore
sizes are larger.
These variations cause formation of highly concentrated bands of sample in
stacking gel and greater resolution of sample components in lower (resolving)
gel.
o Stacking and separation in a discontinuous gel:

o
Buffer compositions control stacking and separation:

o
Glycine equilibria:
o
Formation of an ion front:
o
The voltage gradient sharpens the ion boundary:
o
What happens to the proteins?
Proteins have mobilities between those of Gly and Cl-.
o
In separating gel,
o
Glycine mobility increases, becomes greater than protein mobility, but
still slower than Cl-.
o
Protein sample, now in a narrow band, encounters both the increase
in pH and decrease in pore size.
Increase in pH would tend to increase electrophoretic mobility, but
smaller pores decrease mobility.
Relative rate of movement of ions in lower gel is chloride > glycinate >
protein.
Proteins separate based on charge/mass ratio and on size and shape
parameters.

SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis)
a variant of electrophoresis in which the buffers contain SDS, a detergent that
binds to proteins.
Sodium dodecyl sulfate, SDS
CH3(CH2)10CH2-SO4-, Na+







Most proteins bind SDS at a constant ratio of about 1.4 g
SDS/g protein, i.e., about 1 SDS for every 2 amino acid residues,
unfolding the proteins

Sample treatment before running gel included bmercaptoethanol reduction (so no disulfide bonds left) and
heating to ensure complete unfolding and complete separation of
different polypeptide chains

large negative charge resulting from the bound SDS masks the
native charge on the protein, so that all proteins have essentially
the same charge to mass ratio (very negative), and same shape
("random coil") so

rate of movement in the electric field (toward the + pole
because of – charge on sulfates) depends only on the molecular
weight of individual polypeptide chains (which travel separately)

Protein mobility INVERSELY proportional to the log of
the MASS of individual polypeptide chains, and net charge of
protein itself hardly makes any difference at all.

SDS-PAGE often used to
o o ESTIMATE PURITY (number of stained or
radioactive or fluorescent bands on the gel) and to
o o DETERMINE MOLECULAR WEIGHT of
INDIVIDUAL POLYPEPTIDE SUBUNITS of proteins
(using standards of known polypeptide chain mass)
o o Purification of small amounts of polypeptide for
sequence analysis
Estimating protein molecular weight from SDS gel electrophoresis
a) Diagram of a stained SDS gel: standards of known molecular weight (lane 1)
and pure protein of unknown M.W. in lane 2
b) "standard curve" (calibration) to relate M.W. to mobility on THIS GEL
o
o
o
o
Elution profile
from an anion
exchange resin
(binds negatively
charged proteins)
Proteins were
eluted by
increasing NaCl
concentration in
the eluting buffer.
Total protein was
measured by
determining the
absorbance at 280
nm.
In order to "assay"
(identify) the fatty
acid-binding
proteins, they were
o
o
o

labeled by binding
radioactive fatty
acids
(CPM=counts per
minute - gray
shading).
Purity of each
peak was assessed
using SDS-PAGE
(insert/overlay).
There are two
nearly pure
proteins that bind
fatty acids.
The two proteins
were obtained in
pure form
following one
additional step
(not shown).
Western blotting is an immunological technique for detecting a specific protein in
a mixture separated by gel electrophoresis, using antibodies specific for that
protein to detect it on the gel.
Isoelectric Focusing

separation based on differences in ISOELECTRIC POINT (pI) (so based on
CHARGE DIFFERENCES)




Isoelectric Focusing
pH gradient set up first (using purchased
mixture of ampholytes, different
molecules designed to have range of pIs,
which are first electrophoresed on the
gel to form the pH gradient)
Mixture of molecules (proteins) is then
applied, electric field is turned on, and
each protein moves to the position (pH)
at which its net charge is zero, i.e., its pI.
Two-dimensional
Electrophoresis
isoelectric
focusing in first
dimension,
followed by SDSPAGE at 90o to
that (2nd
dimension)
Ultracentrifugation


Molecular Weight and Shape = fundamental physical properties of a protein.
Estimates of molecular weight can be obtained using SDS-PAGE or gel
filtration, as described above.


One very useful technique for measuring molecular weight and shape is
centrifugation.
A particle that's subjected to a centrifugal field by being spun in a centrifuge is
subjected to a force,
where m is the mass of the particle, r is the distance of the particle from
the center of rotation, and w is the angular velocity.
= buoyancy factor, which accounts for the fact that particle is
buoyed up by the surrounding solvent of density r (g/ml).
is the specific volume of the particle (ml/g) (= 1/density of the particle).
If = r then the particle will not move.


Movement of particle through the solvent is resisted by a frictional coefficient, f,
that depends on the shape of the particle.
Frictional coefficient is an important factor in any transport process, such as
centrifugation or gel filtration.
A spherical particle has f = 1.0, whereas a cigar-shaped or cylindrically-shaped
particle will have f > 1.0.
Movement of any particle under the influence of a centrifugal field is
characterized by its sedimentation coefficient, S, which is directly proportional to
its molecular mass, M, and inversely proportional to f.
, where N is Avogadro's number.

Ultracentrifugation is used in two ways to characterize proteins:
In sedimentation equilibrium
experiments, the centrifuge is operated at
a relative low speed so that the forces of
sedimentation and diffusion balance and
the protein distributes in the centrifuge
cell in a manner proportional to its
molecular weight.
In sedimentation velocity experiments,
the centrifuge is operated at maximal
speed, which causes the protein to
sediment to the bottom of the tube. The
rate at which the boundary moves gives
S, which when combined with M gives f,
a measure of the shape of the protein.
Spectroscopic Methods

Ultraviolet-visible spectroscopy (uv-vis frequency range)
o The principles of absorption spectroscopy. (a) Electronic and
vibrational transitions in a diatomic molecule. (b) The electromagnetic
spectrum.
Introduction
Many compounds absorb ultraviolet (UV) or visible (Vis.) light. The diagram below
shows a beam of monochromatic radiation of radiant power P0, directed at a sample
solution. Absorption takes place and the beam of radiation leaving the sample has radiant
power P.
The amount of radiation absorbed may be
measured in a number of ways:
Transmittance, T = P / P0
% Transmittance, %T = 100 T
Absorbance,
A = log10 P0
/P
A = log10 1 / T
A = log10
100 / %T
A = 2 - log10
%T
The last equation, A = 2 - log10 %T , is worth remembering because it allows you to
easily calculate absorbance from percentage transmittance data.
The relationship between absorbance and transmittance is illustrated in the following
diagram:
So, if all the light passes through a solution without any absorption, then absorbance is
zero, and percent transmittance is 100%. If all the light is absorbed, then percent
transmittance is zero, and absorption is infinite.
The Beer-Lambert Law
Now let us look at the Beer-Lambert law and explore it's significance. This is important
because people who use the law often don't understand it - even though the equation
representing the law is so straightforward:
A=αlc
Where A is absorbance (no units, since A = log10 P0 / P )
α is the molar absorbtivity with units of L mol-1 cm-1
l is the path length of the sample - that is, the path length of the cuvette in which the sample is contained. We will express this
measurement in centimetres.
c is the concentration of the compound in solution, expressed in mol L-1
The reason why we prefer to express the law with this equation is because absorbance is
directly proportional to the other parameters, as long as the law is obeyed. We are not
going to deal with deviations from the law.
Let's have a look at a few questions...
Question : Why do we prefer to express the Beer-Lambert law using absorbance as a
measure of the absorption rather than %T ?
Answer : To begin, let's think about the equations...
A=αlc
%T = 100 P/P0
Now, suppose we have a solution of copper sulphate (which appears blue because it has
an absorption maximum at 600 nm). We look at the way in which the intensity of the
light (radiant power) changes as it passes through the solution in a 1 cm cuvette. We will
look at the reduction every 0.2 cm as shown in the diagram below. The Law says that
the fraction of the light absorbed by each layer of solution is the same. For our
illustration, we will suppose that this fraction is 0.5 for each 0.2 cm "layer" and calculate
the following data:
Path length / cm
0
0.2 0.4 0.6
0.8
1.0
%T
100
50
25
12.5
6.25
3.125
Absorbance
0
0.3
0.6
0.9
1.2
1.5
A = tells us that absorbance depends on the total quantity of the absorbing compound in
the light path through the cuvette. If we plot absorbance against concentration, we get a
straight line passing through the origin (0,0).
Note that the Law is not
obeyed at high
concentrations. This
deviation from the Law
is not dealt with here.
The linear relationship between concentration and absorbance is both simple and
straightforward, which is why we prefer to express the Beer-Lambert law using
absorbance as a measure of the absorption rather than %T.
Theoretical principles
Introduction
Many molecules absorb ultraviolet or visible light. The absorbance of a solution increases
as attenuation of the beam increases. Absorbance is directly proportional to the path
length, b, and the concentration, c, of the absorbing species. Beer's Law states that
A = αlc where α is a constant of proportionality, called the absorbtivity.
Different molecules absorb radiation of different wavelengths. An absorption spectrum
will show a number of absorption bands corresponding to structural groups within the
molecule. For example, the absorption that is observed in the UV region for the carbonyl
group in acetone is of the same wavelength as the absorption from the carbonyl group in
diethyl ketone.
Electronic transitions
1. The absorption of UV or visible radiation corresponds to the excitation of outer
electrons.
When an atom or molecule absorbs energy, electrons are promoted from their ground
state to an excited state. In a molecule, the atoms can rotate and vibrate with respect to
each other. These vibrations and rotations also have discrete energy levels, which can be
considered as being packed on top of each electronic level.
Absorbing species
Absorption of ultraviolet and visible radiation in organic molecules is restricted to certain
functional groups (chromophores) that contain valence electrons of low excitation
energy. The spectrum of a molecule containing these chromophores is complex. This is
because the superposition of rotational and vibrational transitions on the electronic
transitions gives a combination of overlapping lines. This appears as a continuous
absorption band.
Transitions

FLUORESCENCE SPECTROSCOPY
o : Fluorescence. (a) The principle of fluorescence. (b)
Absorption and fluorescence emission spectra of tyrosine.
In most cases, molecules raised to an excited electronic state
by absorption of radiant energy return to ground state by
radiationless transfer of the excitation energy to the
surrounding molecules in the form of heat.
o
o
o
o
o
o
o
Sometimes an excited-state molecule will lose only
part of its energy by transfer (yellow arrow below), and
will re-radiate the larger part as light (green arrow below).
That emitted light is fluorescence.
o Since energy of emitted light is always lower than
energy of absorbed light, fluorescence emission is always
at a longer wavelength than wavelength of the exciting
(absorbed) light (Fig. 6A.4(b) below).
o
terminology
Fluorophore = a molecule that absorbs light but then
returns to the ground state by emitting some of the light as
a photon rather than losing all the energy as heat
o wavelength and intensity of emitted light both very
sensitive to the environment of the fluorophore (e.g.,
hydrophobic vs. aqueous environment can shift emission
spectrum)
o measurements very sensitive so can detect small
amounts of protein or other fluorophore
o Fluorophores in proteins
Trp (maximum wavelength of fluorescence emission
(lmax ~340 nm) is the strongest source of intrinsic
fluorescence in proteins without fluorescent prosthetic
groups, but tyrosine also contributes to intrinsic
fluorescence (see Fig. 6a.4(b) above.

o

o

Some ligands and prosthetic groups are
fluorescent, e.g. the chromophore in green
fluorescent protein
USES of fluorescence spectroscopy -- examples:
 
detect conformational changes
 
e.g. during protein folding (environment
of chromophore affects lmax and intensity of
Trp fluorescence; the more hydrophobic the
environment, e.g. as Trp residues get buried
in the interior of the protein during folding,
the shorter the wavelength of maximum
fluorescence emission)
 
detect and quantitate ligand binding
NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY
(microwave, i.e. radio, frequency range)
o Basis: A spinning charged particle (in this case, a nucleus) behaves as a
magnet, and can interact with an externally imposed magnetic field such
that absorbance of electromagnetic radiation of appropriate energy (in the
microwave, i.e. radio, frequency range) can flip the spin.
o Nuclei used in biochemical studies include 1H (proton NMR), 2H, 13C,
14
N, 17O, 31P, and 19F (in 19F-Tyr).
o To get an NMR spectrum (in ppm), you "sit" on a magnetic field strength
and change the radio frequency to get resonance.
 The type of nucleus you're observing, but also the molecular
structural environment of the nucleus (including its solvent and
surroundings in 3 dimensional space) affect the width and position
(position = "chemical shift") of the NMR signal (peak) for that
nucleus.
 Interaction with a nearby nucleus within the molecule can cause
spin coupling, which is seen as splitting of the NMR signal (double
peak).
 Altering the spin on one nucleus can affect the spin on a nearby
nucleus (< ~5Å away), and for small proteins it is possible by
NMR to do enough distance measurements between nuclei within
the tertiary structure to determine the entire 3-dimensional
structure.
o USES of NMR:
 complete 3-D structure of small proteins in solution (< 25,000
daltons)




conformational changes (e.g., during folding)
determination of pKa of an ionizable group, e.g. His
follow ligand binding
dynamics (motion in solution), e.g. Tyr and Phe ring flips

o
ABSORPTION SPECTROSCOPY
 terminology:
Absorption = transfer of energy from a photon (light) to a
molecule
Chromophore = a molecule or a group on a molecule that absorbs
light
 Chromophores in proteins include
 the peptide bond (maximum wavelength of absorbed light,
lmax, ~220 nm, "far" uv)
 aromatic a.a. residues (lmax ~280 nm for Trp, "near" uv;
aromatics also absorb ~220 nm)
 some prosthetic groups (tightly bound non-amino acid
components in proteins, e.g., the heme in hemoglobin and
myoglobin is red -- it absorbs visible light.)
 USES of absorbance spectroscopy:
 determine concentration (Beer's Law)
 conformational changes (environment of chromophore
affects lmax and absorbance)
 detect and quantitate ligand binding (e.g., O2 binding to
hemoglobin changes absorbance of the heme
Theoretical Principles
Introduction
The term "infra red" covers the range of the electromagnetic spectrum between 0.78 and
"wavenumbers", which have the units cm-1.
wavenumber = 1 / wavelength in centimeters
It is useful to divide the infra red region into three sections; near, mid and far infra red;
Region Wavelength range) Wavenumber range (cm-1)
Near
0.78 - 2.5
12800 - 4000
Middle
2.5 - 50
4000 - 200
Far
50 -1000
200 - 10
The most useful I.R. region lies between 4000 - 670cm-1.
Theory of infra red absorption
IR radiation does not have enough energy to induce electronic transitions as seen with
UV. Absorption of IR is restricted to compounds with small energy differences in the
possible vibrational and rotational states.
For a molecule to absorb IR, the vibrations or rotations within a molecule must cause a
net change in the dipole moment of the molecule. The alternating electrical field of the
radiation (remember that electromagnetic radation consists of an oscillating electrical
field and an oscillating magnetic field, perpendicular to each other) interacts with
fluctuations in the dipole moment of the molecule. If the frequency of the radiation
matches the vibrational frequency of the molecule then radiation will be absorbed,
causing a change in the amplitude of molecular vibration.
Molecular rotations
Rotational transitions are of little use to the spectroscopist. Rotational levels are
quantized, and absorption of IR by gases yields line spectra. However, in liquids or
solids, these lines broaden into a continuum due to molecular collisions and other
interactions.
Molecular vibrations
The positions of atoms in a molecules are not fixed; they are subject to a number of
different vibrations. Vibrations fall into the two main catagories of stretching and
bending.
Stretching: Change in inter-atomic distance along bond axis
Bending: Change in angle between two bonds. There are four types of bend:




Rocking
Scissoring
Wagging
Twisting
Vibrational coupling
In addition to the vibrations mentioned above, interaction between vibrations can occur
(coupling) if the vibrating bonds are joined to a single, central atom. Vibrational coupling
is influenced by a number of factors;





Strong coupling of stretching vibrations occurs when there is a common atom
between the two vibrating bonds
Coupling of bending vibrations occurs when there is a common bond between
vibrating groups
Coupling between a stretching vibration and a bending vibration occurs if the
stretching bond is one side of an angle varied by bending vibration
Coupling is greatest when the coupled groups have approximately equal energies
No coupling is seen between groups separated by two or more bonds
Infrared Spectrophotometry
In the emission spectra of hydrogen, helium, and mercury, the colored lines are the light
energy emitted when an electron falls from a higherenergy state down to a lower energy
state Electronic absorption spectroscopy in the visible area of the EM spectrum measures
the energy of the reverse process, i.e., the wavelength of light required to promote an
electron to a higher energy orbital. This light is in the visible and UV regions of the
electromagnetic spectrum.
If molecules are excited by photons of infrared light (IR), 600-3500 cm^-1, the electrons
in the covalent bonds of those molecules will vibrate. Different types of bonds (C-C, CO, C-H, C=O, etc.) vibrate at distinct IR frequencies. The absorption of infrared light
results in bending, stretching, scissoring, rocking, and other vibrations of the bonds. The
energy of the vibration depends on the type of vibrational mode (bending, rocking, etc.),
the mass of the atoms across the bond, and the strength of the bond.An instrument which
measures the absorption of infrared light by molecules is called an Infrared
Spectrophotometer. For example, C-O stretching vibrations appear within the same
region of the spectrum, , regardless of what else are bonded to those atoms .
C=O stretches occur at higher frequencies, around 1600-1700 cm^-1, because the bond is
stronger. Due to this characteristic behavior, one can usually get a rough idea of the types
of atoms and bonds present in a molecule by looking at the wavelengths (frequencies) of
the IR absorption bands.
The IR spectrum is divided roughly into two sections. The area from 3500-1500 cm^-1 is
called the functional group area; it identifies the presence of alkanes, alkenes, alkynes,
aldehydes, alcohols, etc.
The rest of the spectrum is known as the fingerprint region. The peaks in this part are
fairly unique to the substance being analyzed and a peak-by-peak correspondence to a
known spectrum confirms identification.
Group
-CH3
-CH2-O-H
Principal Transmittance Bands (cm^-1)
2962
2872
1460
1375
2926
2863
1455
3350+/-
-C-Oaromatic
ring
150
1050-1150
3050+/-50
1601
1500
730
690
This bond can be found in a variety of functional
groups shown below:
ketone
1715
aldehyde
1727
ester 1190-1245
Recycling
Structure
Polymer Name
Symbol
-C=O
polyethylene
terephthalate
Uses
soda
bottles
milk ,
detergent,
high density
bleach,
polyethylene water, and
vinegar
bottles
plumbing
fixtures,
some
water
bottles,
polyvinylchloride
glass
cleaner
bottles
(usually
clear)
grocery
and other
shopping
low density
bags,
polyethylene
bread
bags, food
wrap
indooroutdoor
carpeting,
polypropylene
some
yogurt and
margarine
nonrecyclable at this time
containers,
shampoo
and syrup
bottles
toys,
insulated
cups and
polystyrene
containers
(as
styrofoam)
drink
resins, complex boxes and
composites,
squeezable
laminates
catsup
bottles
Introductory theory
Introduction
Few methods of chemical analysis are truly specific to a particular analyte. It is often
found that the analyte of interest must be separated from the myriad of individual
compounds that may be present in a sample. As well as providing the analytical scientist
with methods of separation, chromatographic techniques can also provide methods of
analysis.
Chromatography involves a sample (or sample extract) being dissolved in a mobile phase
(which may be a gas, a liquid or a supercritical fluid). The mobile phase is then forced
through an immobile, immiscible stationary phase. The phases are chosen such that
components of the sample have differing solubilities in each phase. A component which
is quite soluble in the stationary phase will take longer to travel through it than a
component which is not very soluble in the stationary phase but very soluble in the
mobile phase. As a result of these differences in mobilities, sample components will
become separated from each other as they travel through the stationary phase.
Techniques such as H.P.L.C. (High Performance Liquid Chromatography) and G.C. (Gas
Chromatography) use columns - narrow tubes packed with stationary phase, through
which the mobile phase is forced. The sample is transported through the column by
continuous addition of mobile phase. This process is called elution. The average rate at
which an analyte moves through the column is determined by the time it spends in the
mobile phase.
Distribution of analytes between phases
The distribution of analytes between phases can often be described quite simply. An
analyte is in equilibrium between the two phases;
Amobile
Astationary
The equilibrium constant, K, is termed the partition coefficient; defined as the molar
concentration of analyte in the stationary phase divided by the molar concentration of the
analyte in the mobile phase.
The time between sample injection and an analyte peak reaching a detector at the end of
the column is termed the retention time (tR ). Each analyte in a sample will have a
different retention time. The time taken for the mobile phase to pass through the column
is called tM.
A term called the retention factor, k', is often used to describe the migration rate of an
analyte on a column. You may also find it called the capacity factor. The retention factor
for analyte A is defined as;
k'A = t R - tM / tM
t R and tM are easily obtained from a chromatogram. When an analytes retention factor is
less than one, elution is so fast that accurate determination of the retention time is very
difficult. High retention factors (greater than 20) mean that elution takes a very long time.
Ideally, the retention factor for an analyte is between one and five.
We define a quantity called the selectivity factor, α , which describes the separation of
two species (A and B) on the column;
α = k 'B / k 'A
When calculating the selectivity factor, species A elutes faster than species B. The
selectivity factor is always greater than one.
Band broadening and column efficiency
To obtain optimal separations, sharp, symmetrical chromatographic peaks must be
obtained. This means that band broadening must be limited. It is also beneficial to
measure the efficiency of the column.
The Theoretical Plate Model of Chromatography
The plate model supposes that the chromatographic column is contains a large number of
separate layers, called theoretical plates. Separate equilibrations of the sample between
the stationary and mobile phase occur in these "plates". The analyte moves down the
column by transfer of equilibrated mobile phase from one plate to the next.
It is important to remember that the plates do not really exist; they are a figment of
the imagination that helps us understand the processes at work in the column.They also
serve as a way of measuring column efficiency, either by stating the number of
theoretical plates in a column, N (the more plates the better), or by stating the plate
height; the Height Equivalent to a Theoretical Plate (the smaller the better).
If the length of the column is L, then the HETP is
HETP = L / N
The number of theoretical plates that a real column possesses can be found by examining
a chromatographic peak after elution;
where w1/2 is the peak width at half-height.
As can be seen from this equation, columns behave as if they have different numbers of
plates for different solutes in a mixture.
The Rate Theory of Chromatography
A more realistic description of the processes at work inside a column takes account of the
time taken for the solute to equilibrate between the stationary and mobile phase (unlike
the plate model, which assumes that equilibration is infinitely fast). The resulting band
shape of a chromatographic peak is therefore affected by the rate of elution. It is also
affected by the different paths available to solute molecules as they travel between
particles of stationary phase. If we consider the various mechanisms which contribute to
band broadening, we arrive at the Van Deemter equation for plate height;
HETP = A + B / u + C u
where u is the average velocity of the mobile phase. A, B, and C are factors which
contribute to band broadening.
A - Eddy diffusion
The mobile phase moves through the column which is packed with stationary phase.
Solute molecules will take different paths through the stationary phase at random. This
will cause broadening of the solute band, because different paths are of different lengths.
B - Longitudinal diffusion
The concentration of analyte is less at the edges of the band than at the center. Analyte
diffuses out from the center to the edges. This causes band broadening. If the velocity of
the mobile phase is high then the analyte spends less time on the column, which
decreases the effects of longitudinal diffusion.
C - Resistance to mass transfer
The analyte takes a certain amount of time to equilibrate between the stationary and
mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong
affinity for the stationary phase, then the analyte in the mobile phase will move ahead of
the analyte in the stationary phase. The band of analyte is broadened. The higher the
velocity of mobile phase, the worse the broadening becomes.
Van Deemter plots
A plot of plate height vs. average linear velocity of mobile phase.
Such plots are of considerable use in determining the optimum mobile phase flow rate.
Resolution
Although the selectivity factor, α, describes the separation of band centres, it does not
take into account peak widths. Another measure of how well species have been separated
is provided by measurement of the resolution. The resolution of two species, A and B, is
defined as
Baseline resolution is achieved when R = 1.5
It is useful to relate the resolution to the number of plates in the column, the selectivity
factor and the retention factors of the two solutes;
To obtain high resolution, the three terms must be maximised. An increase in N, the
number of theoretical plates, by lengthening the column leads to an increase in retention
time and increased band broadening - which may not be desirable. Instead, to increase the
number of plates, the height equivalent to a theoretical plate can be reduced by reducing
the size of the stationary phase particles.
It is often found that by controlling the capacity factor, k', separations can be greatly
improved. This can be achieved by changing the temperature (in Gas Chromatography)
or the composition of t α can also be manipulated to improve separations. When α is
close to unity, optimising k' and increasing N is not sufficient to give good separation in a
reasonable time. In these cases, k' is optimised first, and then α is increased by one of the
following procedures:
1. Changing mobile phase composition
2. Changing column temperature
3. Changing composition of stationary phase
4. Using special chemical effects (such as incorporating a species which complexes
with one of the solutes into the stationary phase)
Introduction
Gas chromatography - specifically gas-liquid chromatography - involves a sample being
vapourised and injected onto the head of the chromatographic column. The sample is
transported through the column by the flow of inert, gaseous mobile phase. The column
itself contains a liquid stationary phase which is adsorbed onto the surface of an inert
solid.
Have a look at this schematic diagram of a gas chromatograph:
Instrumental components
Carrier gas
The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium,
argon, and carbon dioxide. The choice of carrier gas is often dependant upon the type of
detector which is used. The carrier gas system also contains a molecular sieve to remove
water and other impurities.
Sample injection port
For optimum column efficiency, the sample should not be too large, and should be
introduced onto the column as a "plug" of vapour - slow injection of large samples causes
band broadening and loss of resolution. The most common injection method is where a
microsyringe is used to inject sample through a rubber septum into a flash vapouriser port
at the head of the column. The temperature of the sample port is usually about 50°C
higher than the boiling point of the least volatile component of the sample. For packed
columns, sample size ranges from tenths of a microliter up to 20 microliters. Capillary
columns, on the other hand, need much less sample. For capillary GC, split/splitless
injection is used. Have a look at this diagram of a split/splitless injector;
The injector can be used in one of two modes; split or splitless. The injector contains a
heated chamber containing a glass liner into which the sample is injected through the
septum. The carrier gas enters the chamber and can leave by three routes (when the
injector is in split mode). The sample vapourises to form a mixture of carrier gas,
vapourised solvent and vapourised solutes. A proportion of this mixture passes onto the
column, but most exits through the split outlet. The septum purge outlet prevents septum
bleed components from entering the column.
Columns
There are two general types of column, packed and capillary (also known as open
tubular). Packed columns contain a finely divided, inert, solid support material
(commonly based on diatomaceous earth) coated with liquid stationary phase. Most
packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm.
Capillary columns have an internal diameter of a few tenths of a millimeter. They can be
one of two types; wall-coated open tubular (WCOT) or support-coated open tubular
(SCOT). Wall-coated columns consist of a capillary tube whose walls are coated with
liquid stationary phase. In support-coated columns, the inner wall of the capillary is lined
with a thin layer of support material such as diatomaceous earth, onto which the
stationary phase has been adsorbed. SCOT columns are generally less efficient than
WCOT columns. Both types of capillary column are more efficient than packed columns.
In 1979, a new type of WCOT column was devised - the Fused Silica Open Tubular
(FSOT) column;
These have much thinner walls than the glass capillary columns, and are given strength
by the polyimide coating. These columns are flexible and can be wound into coils. They
have the advantages of physical strength, flexibility and low reactivity.
Column temperature
For precise work, column temperature must be controlled to within tenths of a degree.
The optimum column temperature is dependant upon the boiling point of the sample. As
a rule of thumb, a temperature slightly above the average boiling point of the sample
results in an elution time of 2 - 30 minutes. Minimal temperatures give good resolution,
but increase elution times. If a sample has a wide boiling range, then temperature
programming can be useful. The column temperature is increased (either continuously or
in steps) as separation proceeds.
Detectors
There are many detectors which can be used in gas chromatography. Different detectors
will give different types of selectivity. A non-selective detector responds to all
compounds except the carrier gas, a selective detector responds to a range of compounds
with a common physical or chemical property and a specific detector responds to a single
chemical compound. Detectors can also be grouped into concentration dependant
detectors and mass flow dependant detectors. The signal from a concentration dependant
detector is related to the concentration of solute in the detector, and does not usually
destroy the sample Dilution of with make-up gas will lower the detectors response. Mass
flow dependant detectors usually destroy the sample, and the signal is related to the rate
at which solute molecules enter the detector. The response of a mass flow dependant
detector is unaffected by make-up gas. Have a look at this tabular summary of common
GC detectors:
Support
Dynamic
Detector
Type
Selectivity
Detectability
gases
range
Flame
ionization
(FID)
Hydrogen
and air
Most organic cpds.
100 pg
107
Thermal
conductivity Concentration Reference
(TCD)
Universal
1 ng
107
Electron
capture
(ECD)
Halides, nitrates,
nitriles, peroxides,
anhydrides,
50 fg
105
Mass flow
Concentration Make-up
organometallics
Nitrogenphosphorus
Mass flow
Hydrogen
and air
Nitrogen, phosphorus 10 pg
106
Flame
photometric Mass flow
(FPD)
Hydrogen
and air
possibly
oxygen
Sulphur, phosphorus,
tin, boron, arsenic,
100 pg
germanium,
selenium, chromium
103
Aliphatics, aromatics,
ketones, esters,
aldehydes, amines,
2 pg
heterocyclics,
organosulphurs,
some organometallics
107
Photoionization
(PID)
Concentration Make-up
Hall
electrolytic Mass flow
conductivity
Hydrogen, Halide, nitrogen,
oxygen
nitrosamine, sulphur
The effluent from the column is mixed with hydrogen and air, and ignited. Organic
compounds burning in the flame produce ions and electrons which can conduct electricity
through the flame. A large electrical potential is applied at the burner tip, and a collector
electrode is located above the flame. The current resulting from the pyrolysis of any
organic compounds is measured. FIDs are mass sensitive rather than concentration
sensitive; this gives the advantage that changes in mobile phase flow rate do not affect
the detector's response. The FID is a useful general detector for the analysis of organic
compounds; it has high sensitivity, a large linear response range, and low noise. It is also
robust and easy to use, but unfortunately, it destroys the sample.
DNA technology
Determining the molecular sequence of DNA that makes up the genome of different
organisms is an international scientific goal, several laboratories are participating
worldwide in this task
Recombinant DNA Technology
Techniques for
- Isolation
- Digestion
- Fractionation
- Purification of the TARGET fragment
- Cloning into vectors
- Transformation of host cell and selection
- Replication
- Analysis
- Expression of DNA
How do we obtain DNA and how do we manipulate DNA?
Quite straightforward to isolate DNA
For instance, to isolate genomic DNA
1. Remove tissue from organism
2. Homogenise in lysis buffer containing guanidine thiocyanate (denatures
proteins)
3. Mix with phenol/chloroform - removes proteins
4. Keep aqueous phase (contains DNA)
5. Add alcohol (ethanol or isopropanol) to precipitate DNA from solution
6. Collect DNA pellet by centrifugation
7. Dry DNA pellet and resuspend in buffer
8. Store at 4°C
Enzymes that can cut (hydrolyse) DNA duplex at specific sites. Current DNA
technology is totally dependent on restriction enzymes.
Restriction enzymes are endonucleases
Restriction enzymes recognise a specific short nucleotide sequence
This is known as a Restriction Site
The phosphodiester bond is cleaved between specific bases, one on each DNA
strand
Examples of restriction enzymes and the sequences they cleave
Source microorganism
Enzyme
Arthrobacter luteus
Alu I
Bacillus amyloiquefaciens H
Recognition Site
Ends produced
AG CT
Blunt
Bam HI
G GATCC
Sticky
Escherichia coli
Eco RI
G AATTC
Sticky
Haemophilus gallinarum
Hga I
GACGC(N)5
Sticky
Haemophilus infulenzae
Hind III
A AGCTT
Sticky
Providencia stuartii 164
Pst I
CTGCA G
Sticky
Nocardia otitiscaviaruns
Not I
GC GGCCGC Sticky
DNA fractionation
Separation of DNA fragments in order to isolate and analyse DNA cut by
restriction enzymes
Electrophoresis
Linear DNA fragments of different sizes are resolved according to their size
through gels made of polymeric materials such as polyacrylamide and agarose.
For instance, agarose is a polysaccharide derived from seaweed - and gels formed
from between 0.5% to 2% (mass/volume i.e. 0.5 to 2.0g agarose/100 ml of
aqueous buffer) can be used to separate (resolve) most sizes of DNA
DNA is electrophoresed through the agarose gel from the cathode (negative) to
the anode (positive) when a voltage is applied, due to the net negative charge
carried on DNA
When the DNA has been electrophoresed, the gel is stained in a solution
containing the chemical ethidium bromide. This compound binds tightly to DNA
(DNA chelator) and fluoresces strongly under UV light - allowing the
visualisation and detection of the DNA.
Analysing complex nucleic acid mixtures (DNA or RNA)
The total cellular DNA of an organism (genome) or the cellular content of RNA
are complex mixtures of different nucleic acid sequences. Restriction digest of a
complex genome can generate millions of specific restriction fragments and there
can be several fragments of exactly the same size which will not be separated
from each other by electrophoresis.
Techniques have been devised to identify specific nucleic acids in these complex
mixtures


Southern blotting - DNA
Northern blotting - RNA
These techniques are not to be confused with Western blotting, which is used to
analyse PROTEINS which have been immobilised on nitrocellulose/nylon filters.
Proteins which have been separated by polyacrylamide gel electrophoresis
(PAGE) are transferred to nitrocellulose/nylon filters and the filter is probed with
antibodies to detect the specific protein - similar to the method used for
expression library screening.
Southern blotting
This technique, devised by Ed Southern in 1975, is a commonly used method for
the identification of DNA fragments that are complementary to a know DNA
sequence. Southern hybridisation, also called Southern blotting, allows a
comparison between the genome of a particular organism and that of an available
gene or gene fragment (the probe). It can tell us whether an organism contains a
particular gene, and provide information about the organisation and restriction
map of that gene.
In Southern blotting, chromosomal DNA is isolated from the organism of
interest, and digested to completion with a restriction endonuclease enzyme. The
restriction fragments are then subjected to electrophoresis on an agarose gel,
which separates the fragments on the basis of size.
DNA fragments in the gel are denatured (i.e. separated into single strands) using
an alkaline solution. The next step is to transfer fragments from the gel onto
nitrocellulose filter or nylon membrane. This can be performed by
electrotransfer (electrophoresing the DNA out of the gel and onto a
nitrocellulose filter), but is more typically performed by simple capillary action.
In this system, the denatured gel is placed onto sheet(s) of moist filter paper and
immersed in a buffer reservoir. A nitrocellulose membrane is laid over the gel,
and a number of dry filter papers are placed on top of the membrane. By capillary
action, buffer moves up through the gel, drawn by the dry filter paper. It carries
the single-stranded DNA with it, and when the DNA reaches the nitrocellulose it
binds to it and is immobilised in the same position relative to where it had
migrated in the gel.
The DNA is bound irreversibly to the filter/membrane by baking at high
temperature (nitrocellulose) or cross-linking through exposure to UV light
(nylon).
The final step is to immerse the membrane in a solution containing the probe either a DNA (cDNA clone, genomic fragment, oligonucleotide) or RNA probe
can be used. This is DNA hybridisation - in other words the target DNA and the
probe DNA/RNA form a 'hybrid' because they are complementary sequences and
so can bind to each other. The probe is usually radioactively labelled with 32P,
often by removal of the 5' phosphate of the probe with alkaline phosphatase, and
replacement with a radiolabelled phosphate using α -[32P]ATP and polynucleotide
kinase. The membrane is washed to remove non-specifically bound probe (see
washing & stringency conditions), and is then exposed to X-ray film - a process
called autoradiography. At positions where the probe is bound, α -emissions
from the probe cause the X-ray film to blacken. This allows the identification of
the sizes and the number of fragments of chromosomal genes with strong
similarity to the gene or gene fragment used as a probe.
The principle of Southern blotting
What Southern blotting can tell us
1. Whether a particular gene is present and how many copies are present in
the genome of an organism
2. The degree of similarity between the chromosomal gene and the probe
sequence
3. Whether recognition sites for particular restriction endonucleases are
present in the gene. By performing the digestion with different
endonucleases, or with combinations of endonucleases, it is possible to
obtain a restriction map of the gene i.e. an idea of the restriction enzyme
sites in and around the gene- which will assist in attempts to clone the
gene.
4. Whether re-arrangements have occurred during the cloning process
Northern blotting
Northern blotting is a simple extension of Southern blotting - and derives its
name from the earlier technique. It is used to detect cellular RNA rather than
DNA. Initially, it was thought that RNA would not bind efficiently to
nitrocellulose, and other modified materials were synthesised for use as a
membrane. However, it was then shown that when RNA was denatured, that it
would also bind efficiently to nitrocellulose. This means that the RNA has to be
unfolded into a linear strand before it will bind efficiently to nitrocellulose.
Chemicals such as formaldehyde and methylmercuric hydroxide can be used to
denature the RNA - breaking down hydrogen bonding structure in the molecule.
Alkali is not used to denature the RNA - since RNA is degraded under alkaline
conditions.
Isolating RNA
RNA is extracted from the cells of interest - but precautions must be taken to
avoid degradation of the single-stranded RNA by ribonuclease (RNase), which is
found on the skin and on glassware. Wear gloves, use specially treated plastics
and glassware to avoid accidently introducing ribonuclease to extraction prep.
Addition of diethylpyrocabonate (DEPC) inhibits ribonuclease activity and
baking at high temperature destroys ribonuclease activity (only useful for treating
heat resistant equipment, such as glassware).
DNA sequencing: Maxam & Gilbert sequencing
For this method, need to use DNA fragments ~500 nucleotides - for instance by isolating
restriction fragments of the DNA that is to be sequenced. The method is reliable for
sequencing up to ~250-300 nucleotides at a time. The technique requires that the target
DNA is end-labeled (usually radioactively).


Either at the 5' end: add alkaline phosphatase to remove 5' phosphate, and
polynucleotide kinase to add back a radiolabeled phosphate to the 5' OH group
using [α 32P]ATP
Or at the 3' end: add a homopolymeric tail using terminal tranferase and [α
32
P]dATP (or another 32P-labeled deoxyribonucleotide triphosphate)
Both single-stranded (ss) and double-stranded DNA (ds) can be sequenced. If ds DNA is
used, then the label must be removed from one end, so that fragment sizes can be gauged
by the distance to the end of the molecule from one unique label at the other end.
The M&G method involves the chemical degradation of DNA
The process requires addition of chemicals that bring about cleavage of DNA at specific
positions. (The Sanger method involves DNA synthesis).
Either 4 or 5 separate chemical reactions are performed. The reactions are carried
out in two stages:
Stage 1: Specific chemical modification of bases in the DNA
Stage 2: Chemical cleavage of sugar-phosphate backbone at modification site
Stage 1: Specific chemical modification of bases in the DNA
Base
Specific modification
modified
G
Methylation of base with dimethyl sulfate at pH 8.0.
Makes base susceptible to cleavage by alkali
A+G
Treatment with piperidine formate at pH 2.0.
Results in removal of purine bases
C+T
Hydrazine opens pyrimidine rings and causes their removal from DNA
C
In high ionic strength (1.5 M NaCl) only cytosine reacts with hydrazine
Treatment with 1.2 M NaOH at high temperature (90°C) gives strong
cleavage at A, less at C
Stage 2
When bases are modified and destroyed by the treatments in stage 1, piperidine at 90°C is
used to cleave the sugar-phosphate backbone at the site.
A>C
For example:
The 'trick' in the reaction is to limit incubation times with base-modifying reagents
and/or the concentrations of reagents used, so that a ladder of progressively longer
molecules is generated in the M&G sequencing reactions.
The differently sized/labelled fragments are separated by polyacrylamide gel
electorphoresis (PAGE)
Limitations: Resolving power of the gel. A typical gel is composed of 6% acrylamide
(6g/100ml). By altering the % of the acrylamide, shorter fragments (use higher %) or
longer fragments (use lower %) can be resolved. Samples can also be electrophoresed for
different lengths of time to resolve fragments of different sizes.
Dangers: The chemicals used destroy DNA - they are mutagens
Sanger dideoxy sequencing
A DNA synthesis reaction. Needs ss DNA as the template. If only ds DNA is available,
can be treated with alkali to separate it into single strands (denature it). Rather more
bases can usually be read by the Sanger technique: ~500 vs ~300 by the M&G
technique.
The Sanger method
The reaction requires an oligonucleotide primer (typically 16-17 nucleotides long). The
primer is annealed to the ssDNA template. Four separate synthesis reactions are set up
for each DNA template/oligo primer.
Method uses 4 different dideoxyribonucleotide triphosphates (ddNTPs), one for each
reaction mix
Remember: for DNA polymerase to continue synthesising the DNA strand, it requires a
free -OH group at the 3' position of the sugar. Look carefully - this is missing from the
dideoxyribonucleotide.
This is the chemistry behind the technique: the phosphodiester bond that forms between
successive nucleotides in a DNA chain requires a 3' hydroxyl group. The oligonucleotide
primer provides the first free -OH group and each successive nucleotide that is added
provides the next. ddNTPs differ from ordinary dNTPs in that they have a hydrogen
(rather than a hydroxy) on the 3' position of the sugar. Once a ddNTP is incorporated,
the nucleotide chain can't be extended from that point.
In each of 4 reaction mixtures the following reagents are mixed
1. Template ssDNA
2. Primer
3. Each of the 4 dNTPs (dATP, dCTP, dTTP, dGTP)
4. DNA polymerase (Klenow fragment or T7 DNA polymerase)
5. One of the 4 possible ddNTPs:
'G mix': add ddGTP
'A mix': add ddATP
'T mix': add ddTTP
'C mix': add ddCTP
Sanger chain termination technique
The newly synthesised strand is labeled with a radionucleotide.
Either the primer is 5' end-labelled with 32P or a radiolabeled deoxyribonucleotide is
incorporated in the growing chain ([α 35S]dATP is popularly used).
In each of the 4 mixes, the ratio of dNTP:ddNTP is critical. The ratio is usually ~100:1.
This results in a reasonably random termination of chains of DNA at different positions.
Each of the 4 synthesis mixes will contain a series of differently sized radiolabeled DNA
fragments, in each mixture all will terminate at the same type of nucleotide. The length is
dependent on how far the nucleotide chain is extended before a ddNTP is incorporated.
Chain termination at different G residues: the 'G mix' reaction
Like M&G sequencing, the reactions are loaded into 4 adjacent lanes on a long gel for
resolving the different sized fragments by PAGE.
After electrophoresis the gel is dried and exposed to X-ray film and the sequence read
from the bottom to the top of the gel