ANALYSIS OF PROTEINS

PD 203 Pharmacognosy & Phytopharmaceuticals

 Introduction

• Proteins are polymers of amino acids. Twenty different types of amino acids occur
naturally in proteins.
• Proteins differ from each other according to the type, number and sequence of amino
acids that make up the polypeptide backbone.
• As a result they have different molecular structures, nutritional attributes and
physiochemical properties.
• Proteins are important constituents of foods for a number of different reasons. They
are a major source of energy, as well as containing essential amino-acids, such as
lysine, tryptophan, methionine, leucine, isoleucine and valine, which are essential to
human health, but which the body cannot synthesize.
• Proteins are also the major structural components of many natural foods, often
determining their overall texture, e.g., tenderness of meat or fish products.
• Isolated proteins are often used in foods as ingredients because of their unique
functional properties, i.e., their ability to provide desirable appearance, texture or
stability.
• Typically, proteins are used as gelling agents, emulsifiers, foaming agents and
thickeners.
• Many food proteins are enzymes which are capable of enhancing the rate of certain
biochemical reactions.
• These reactions can have either a favorable or detrimental effect on the overall
properties of foods. Food analysts are interested in knowing the total concentration,
type, molecular structure and functional properties of the proteins in foods.
DETERMINATION OF OVERALL
PROTEIN CONCENTRATION
 Kjeldahl method

• The Kjeldahl method was developed in 1883 by a brewer called Johann Kjeldahl. A
food is digested with a strong acid so that it releases nitrogen which can be
determined by a suitable titration technique.
• The amount of protein present is then calculated from the nitrogen concentration of
the food. The same basic approach is still used today, although a number of
improvements have been made to speed up the process and to obtain more
accurate measurements.
• It is usually considered to be the standard method of determining protein
concentration. Because the Kjeldahl method does not measure the protein content
directly a conversion factor (F) is needed to convert the measured nitrogen
concentration to a protein concentration.
 Principles

• Digestion
• The food sample to be analyzed is weighed into a digestion flask and then digested
by heating it in the presence of sulfuric acid (an oxidizing agent which digests the
food), anhydrous sodium sulfate (to speed up the reaction by raising the boiling
point) and a catalyst, such as copper, selenium, titanium, or mercury (to speed up
the reaction).
• Digestion converts any nitrogen in the food (other than that which is in the form of
nitrates or nitrites) into ammonia, and other organic matter to C02 and H20.
Ammonia gas is not liberated in an acid solution because the ammonia is in the
form of the ammonium ion (NH4+) which binds to the sulfate ion (SO42-) and thus
remains in solution:
N(food)  (NH4)2SO4 (1)
• Neutralization
• After the digestion has been completed the digestion flask is connected to a recieving flask by a
tube. The solution in the digestion flask is then made alkaline by addition of sodium hydroxide,
which converts the ammonium sulfate into ammonia gas:
(NH4)2SO4 + 2 NaOH  2NH3 + 2H2O + Na2SO4 (2)
• The ammonia gas that is formed is liberated from the solution and moves out of the digestion flask
and into the receiving flask - which contains an excess of boric acid. The low pH of the solution in
the receiving flask converts the ammonia gas into the ammonium ion, and simultaneously converts
the boric acid to the borate ion:
NH3 + H3BO3 (boric acid)  NH4+ + H2BO3- (borate ion) (3)
• Titration
• The nitrogen content is then estimated by titration of the ammonium borate
formed with standard sulfuric or hydrochloric acid, using a suitable indicator
to determine the end-point of the reaction.
H2BO3- + H+  H3BO3 (4)
The concentration of hydrogen ions (in moles) required to reach the end-
point is equivalent to the concentration of nitrogen that was in the original
food (Equation 3). The following equation can be used to determine the
nitrogen concentration of a sample that weighs m grams using a xM HCl
acid solution for the titration:

Where vs and vb are the titration volumes of the sample and blank, and 14g is the molecular weight of
nitrogen N. A blank sample is usually ran at the same time as the material being analyzed to take into
account any residual nitrogen which may be in the reagents used to carry out the analysis. Once the
nitrogen content has been determined it is converted to a protein content using the appropriate
conversion factor: %Protein = F� %N.
Advantages and Disadvantages

• Advantages. The Kjeldahl method is widely used internationally and is still the
standard method for comparison against all other methods. Its universality, high
precision and good reproducibility have made it the major method for the
estimation of protein in foods.
• Disadvantages. It does not give a measure of the true protein, since all nitrogen in
foods is not in the form of protein. Different proteins need different correction
factors because they have different amino acid sequences. The use of
concentrated sulfuric acid at high temperatures poses a considerable hazard, as
does the use of some of the possible catalysts The technique is time consuming to
carry-out.
Enhanced Dumas method
• Recently, an automated instrumental technique has been
developed which is capable of rapidly measuring the
protein concentration of food samples. This technique is
based on a method first described by a scientist called
Dumas over a century and a half ago. It is beginning to
compete with the Kjeldahl method as the standard method
of analysis for proteins for some foodstuffs due to its
rapidness.
General Principles
• A sample of known mass is combusted in a high temperature (about 900
o
C) chamber in the presence of oxygen. This leads to the release of CO2,
H2O and N2. The CO2 and H2O are removed by passing the gasses over
special columns that absorb them. The nitrogen content is then measured
by passing the remaining gasses through a column that has a thermal
conductivity detector at the end. The column helps separate the nitrogen
from any residual CO2 and H2O that may have remained in the gas stream.
The instrument is calibrated by analyzing a material that is pure and has a
known nitrogen concentration, such as EDTA (= 9.59%N). Thus the signal
from the thermal conductivity detector can be converted into a nitrogen
content. As with the Kjeldahl method it is necessary to convert the
concentration of nitrogen in a sample to the protein content, using suitable
conversion factors which depend on the precise amino acid sequence of
the protein.
Advantages and Disadvantages
• Advantages: It is much faster than the Kjeldahl method (under 4 minutes per
measurement, compared to 1-2 hours for Kjeldahl). It doesn't need toxic
chemicals or catalysts. Many samples can be measured automatically. It is
easy to use.
• Disadvantages: High initial cost. It does not give a measure of the true protein,
since all nitrogen in foods is not in the form of protein. Different proteins need
different correction factors because they have different amino acid sequences.
The small sample size makes it difficult to obtain a representative sample.
. METHODS USING UV-VISIBLE
SPECTROSCOPY
UV-visible spectroscopy

• A number of methods have been devised to measure protein concentration, which are
based on UV-visible spectroscopy.
• These methods use either the natural ability of proteins to absorb (or scatter) light in
the UV-visible region of the electromagnetic spectrum, or they chemically or physically
modify proteins to make them absorb (or scatter) light in this region.
• The basic principle behind each of these tests is similar.
• First of all a calibration curve of absorbance (or turbidity) versus protein concentration
is prepared using a series of protein solutions of known concentration.
• The absorbance (or turbidity) of the solution being analyzed is then measured at the
same wavelength, and its protein concentration determined from the calibration curve.
• The main difference between the tests are the chemical groups which are responsible
for the absorption or scattering of radiation, e.g., peptide bonds, aromatic side-groups,
basic groups and aggregated proteins.
Principles
• Direct measurement at 280nm
• Tryptophan and tyrosine absorb ultraviolet light strongly at 280 nm.
• The tryptophan and tyrosine content of many proteins remains fairly constant, and so the
absorbance of protein solutions at 280nm can be used to determine their concentration.
• The advantages of this method are that the procedure is simple to carry out, it is
nondestructive, and no special reagents are required.
• The major disadvantage is that nucleic acids also absorb strongly at 280 nm and could
therefore interfere with the measurement of the protein if they are present in sufficient
concentrations. Even so, methods have been developed to overcome this problem, e.g., by
measuring the absorbance at two different wavelengths.
• Biuret Method
• A violet-purplish color is produced when cupric ions (Cu 2+) interact with peptide bonds
under alkaline conditions.
• The biuret reagent, which contains all the chemicals required to carry out the analysis, can
be purchased commercially.
• It is mixed with a protein solution and then allowed to stand for 15-30 minutes before the
absorbance is read at 540 nm.
• The major advantage of this technique is that there is no interference from materials that
adsorb at lower wavelengths, and the technique is less sensitive to protein type because it
utilizes absorption involving peptide bonds that are common to all proteins, rather than
specific side groups.
• However, it has a relatively low sensitivity compared to other UV-visible methods.
• Procedure
• Reagent
• A formula for biuret reagent is (per liter final volume) 9 gm Sodium potassium tartrate (f.w. 282.22), 3 gm Copper sulfate x 5
H2O (f.w. 249.68), 5 gm Potassium iodide (166.0), all dissolved in order in 400 ml 0.2 M NaOH (f.w. 40.0) before bringing to
final volume. The volume can be scaled up or scaled down of course. Discard if a black precipitate forms.
• Assay
• Volumes sample, reagent can be scaled up/down and/or volume ratios varied, as with any assay.
• Warm up the spectrophotometer 15 min. before use.
• Prepare standards from bovine serum albumin, preferably calibrated using absorbance at 280 nm and the extinction
coefficient. Using 5 ml color reagent to 1 ml sample a recommended range is 0.5 to 20 mg protein.
• Prepare a reference tube with 1 ml buffer.
• If possible, dilute unknowns to an estimated 1 to 10 mg/ml with buffer; a range of dilutions should be used if the actual
concentration cannot be estimated.
• Use 1 ml sample per assay tube
• Add 9 ml Biuret reagent to each tube, vortex immediately, and let stand 20 min.
• Read at 550 nm.
• Analysis
• Prepare a standard curve of absorbance versus micrograms protein (or vice
versa), and determine amounts from the curve. Determine concentrations of
original samples from the amount protein, volume/sample, and dilution factor,
if any.
• Lowry Method
• The Lowry method combines the biuret reagent with another reagent (the Folin-Ciocalteau
phenol reagent) which reacts with tyrosine and tryptophan residues in proteins.
• This gives a bluish color which can be read somewhere between 500 - 750 nm depending
on the sensitivity required. There is a small peak around 500 nm that can be used to
determine high protein concentrations and a large peak around 750 nm that can be used to
determine low protein concentrations.
• This method is more sensitive to low concentrations of proteins than the biuret method.
PROTEINS (LOWRY) PROTOCOL
• INTRODUCTION
• The “Lowry Assay: Protein by Folin Reaction” (Lowry et al. , 1951) has been
the most widely used method to estimate the amount of proteins (already in
solution or easily-soluble in dilute alkali) in biological samples. First the
proteins are pre-treated with copper ion in alkali solution, and then the
aromatic amino acids in the treated sample reduce the phosphomolybdate
phosphotungstic acid present in the Folin Reagent. The end product of this
reaction has a blue color. The amount of proteins in the sample can be
estimated via reading the absorbance (at 750 nm) of the end product of the
Folin reaction against a standard curve of a selected standard protein solution
(in our case; Bovine Serum Albumin-BSA- solution).
• PROCEDURE
• • Wear gloves, goggles, lab coat, and work very carefully
• • Take your samples out of -20oC freezer to thaw
• • Prepare the stock BSA solution and dilutions for the standard curve (Table 3.1, prepare freshly)
• • Prepare the Lowry solution by mixing the Sol.A, B, and C.
• • When totally thawed, vortex well and dilute your samples with ddI water if necessary.
• • Work in triplicates
• • Vortex your sample/dilution (or standard) well to mix and transfer 0.5 mL to a 10 mL glass tube.
You can use a 1000 uL pipetman at this step
• • Add 0.7 mL of Lowry Solution to the tubes. You can use a 1000 uL pipet.
• • Cap and vortex briefly to mix (decrease the speed of the vortex to approx. 3-4)
• • Incubate for 0 min at room temp. in dark.
• • At the last 5 minutes, prepare the diluted Folin Reagent.
• • After 20 min of incubation, take the samples out and add 0.1 mL of diluted Folin Reagent to each
tube.
• • Cap and vortex immediately to mix.
• • Incubate once more for 30 min or longer at room temp. in dark.
• • While waiting, turn on the UV-VIS spec to warm up and stabilize
• • After 30 min, vortex the samples briefly
• • Transfer the samples (1.3 mL) to semi-micro disposable cuvettes
• • Working at the Quantitative mode and @750 nm , auto- zero the spec. with 2 semi-micro cuvettes
filled with ddI water. Then record the absorbance values for the standards (standards mode) and
the samples (unknown mode).
• • Prepare the calibration curve from the abs. readings of the standards and calculate the protein
content of the samples in mg BSA/L from this curve.
• Dye binding methods
• A known excess of a negatively charged (anionic) dye is added to a protein solution whose
pH is adjusted so that the proteins are positively charged (i.e. < the isoelectric point).
• The proteins form an insoluble complex with the dye because of the electrostatic attraction
between the molecules, but the unbound dye remains soluble.
• The anionic dye binds to cationic groups of the basic amino acid residues (histidine,
arganine and lysine) and to free amino terminal groups.
• The amount of unbound dye remaining in solution after the insoluble protein-dye complex
has been removed (e.g., by centrifugation) is determined by measuring its absorbance.
• The amount of protein present in the original solution is proportional to the amount of dye
that bound to it:
• dyebound = dyeinitial - dyefree.
• Turbimetric method
• Protein molecules which are normally soluble in solution can be made to precipitate by the
addition of certain chemicals, e.g., trichloroacetic acid. Protein precipitation causes the
solution to become turbid.
• Thus the concentration of protein can be determined by measuring the degree of turbidity.
Advantages and Disadvantages of UV-Visible methods

• Advantages:
• UV-visible techniques are fairly rapid and simple to carry out, and are sensitive to low
concentrations of proteins.
• Disadvantages:
• For most UV-visible techniques it is necessary to use dilute and transparent solutions, which
contain no contaminating substances which absorb or scatter light at the same wavelength as
the protein being analyzed.
• The need for transparent solutions means that most foods must undergo significant amounts of
sample preparation before they can be analyzed, e.g., homogenization, solvent extraction,
centrifugation, filtration, which can be time consuming and laborious.
• In addition, it is sometimes difficult to quantitatively extract proteins from certain types of foods,
especially after they have been processed so that the proteins become aggregated or
covalently bound with other substances. In addition the absorbance depends on the type of
protein analyzed (different proteins have different amino acid sequences).
OTHER INSTRUMENTAL
TECHNIQUES
• There are a wide variety of different instrumental methods available for
determining the total protein content of food materials. These can be divided
into three different categories according to their physicochemical principles:
• (i) measurement of bulk physical properties,
• (ii) measurement of adsorption of radiation, and
• (iii) measurement of scattering of radiation.
• Each instrumental methods has its own advantages and disadvantages, and
range of foods to which it can be applied.
• Principles
• Measurement of Bulk Physical Properties
• Density: The density of a protein is greater than that of most other food components, and so
there is an increase in density of a food as its protein content increases. Thus the protein
content of foods can be determined by measuring their density.
• Refractive index: The refractive index of an aqueous solution increases as the protein
concentration increases and therefore RI measurements can be used to determine the
protein content.
• Measurement of Adsorption of Radiation
• UV-visible: The concentration of proteins can be determined by measuring the absorbance
of ultraviolet-visible radiation (see above).
• Infrared: Infrared techniques can be used to determine the concentration of proteins in food
samples. Proteins absorb IR naturally due to characteristic vibrations (stretching and
bending) of certain chemical groups along the polypeptide backbone. Measurements of the
absorbance of radiation at certain wavelengths can thus be used to quantify the
concentration of protein in the sample. IR is particularly useful for rapid on-line analysis of
protein content. It also requires little sample preparation and is nondestructive. Its major
disadvantages are its high initial cost and the need for extensive calibration.
• Nuclear Magnetic Resonance: NMR spectroscopy can be used to determine the total
protein concentration of foods. The protein content is determined by measuring the area
under a peak in an NMR chemical shift spectra that corresponds to the protein fraction.
• Measurement of Scattering of Radiation
• Light scattering: The concentration of protein aggregates in aqueous solution can be
determined using light scattering techniques because the turbidity of a solution is directly
proportional to the concentration of aggregates present.
• Ultrasonic scattering: The concentration of protein aggregates can also be determined
using ultrasonic scattering techniques because the ultrasonic velocity and absorption of
ultrasound are related to the concentration of protein aggregates present.
• Advantages and Disadvantages
• A number of these instrumental methods have major advantages over the
other techniques mentioned above because they are nondestructive, require
little or no sample preparation, and measurements are rapid and precise. A
major disadvantage of the techniques which rely on measurements of the bulk
physical properties of foods are that a calibration curve must be prepared
between the physical property of interest and the total protein content, and this
may depend on the type of protein present and the food matrix it is contained
within. In addition, the techniques based on measurements of bulk
physicochemical properties can only be used to analyze foods with relatively
simple compositions. In a food that contains many different components whose
concentration may vary, it is difficult to disentangle the contribution that the
protein makes to the overall measurement from that of the other components.
Protein Separation and Characterization
• Protein type is usually determined by separating and isolating the individual proteins
from a complex mixture of proteins, so that they can be subsequently identified and
characterized.
• Proteins are separated on the basis of differences in their physicochemical
properties, such as size, charge, adsorption characteristics, solubility and heat-
stability.
• The choice of an appropriate separation technique depends on a number of factors,
including the reasons for carrying out the analysis, the amount of sample available,
the desired purity, the equipment available, the type of proteins present and the cost.
• Large-scale methods are available for crude isolations of large quantities of proteins,
whereas small-scale methods are available for proteins that are expensive or only
available in small quantities. One of the factors that must be considered during the
separation procedure is the possibility that the native three dimensional structure of
the protein molecules may be altered.
• A prior knowledge of the effects of environmental conditions on protein
structure and interactions is extremely useful when selecting the most
appropriate separation technique.
• Firstly, because it helps determine the most suitable conditions to use to
isolate a particular protein from a mixture of proteins (e.g., pH, ionic strength,
solvent, temperature etc.), and secondly, because it may be important to
choose conditions which will not adversely affect the molecular structure of the
proteins.
METHODS BASED ON DIFFERENT
SOLUBILITY CHARACTERISTICS
Methods Based on Different Solubility Characteristics
• Proteins can be separated by exploiting differences in their solubility in aqueous solutions.
• The solubility of a protein molecule is determined by its amino acid sequence because this
determines its size, shape, hydrophobicity and electrical charge.
• Proteins can be selectively precipitated or solubilized by altering the pH, ionic strength,
dielectric constant or temperature of a solution.
• These separation techniques are the most simple to use when large quantities of sample
are involved, because they are relatively quick, inexpensive and are not particularly
influenced by other food components.
• They are often used as the first step in any separation procedure because the majority of
the contaminating materials can be easily removed.
Salting out

• Proteins are precipitated from aqueous solutions when the salt concentration exceeds a
critical level, which is known as salting-out, because all the water is "bound" to the salts,
and is therefore not available to hydrate the proteins.
• Ammonium sulfate [(NH4)2SO4] is commonly used because it has a high water-solubility,
although other neutral salts may also be used, e.g., NaCl or KCl. Generally a two-step
procedure is used to maximize the separation efficiency. In the first step, the salt is added
at a concentration just below that necessary to precipitate out the protein of interest.
• The solution is then centrifuged to remove any proteins that are less soluble than the
protein of interest. The salt concentration is then increased to a point just above that
required to cause precipitation of the protein. This precipitates out the protein of interest
(which can be separated by centrifugation), but leaves more soluble proteins in solution.
• The main problem with this method is that large concentrations of salt contaminate the
solution, which must be removed before the protein can be resolubilzed, e.g., by dialysis or
ultrafiltration.
Isoelectric Precipitation
• The isoelectric point (pI) of a protein is the pH where the net charge on the
protein is zero. Proteins tend to aggregate and precipitate at their pI because
there is no electrostatic repulsion keeping them apart. Proteins have different
isoelectric points because of their different amino acid sequences (i.e., relative
numbers of anionic and cationic groups), and thus they can be separated by
adjusting the pH of a solution. When the pH is adjusted to the pI of a particular
protein it precipitates leaving the other proteins in solution.
Solvent Fractionation

• The solubility of a protein depends on the dielectric constant of the solution that surrounds it
because this alters the magnitude of the electrostatic interactions between charged groups.
• As the dielectric constant of a solution decreases the magnitude of the electrostatic
interactions between charged species increases.
• This tends to decrease the solubility of proteins in solution because they are less ionized, and
therefore the electrostatic repulsion between them is not sufficient to prevent them from
aggregating.
• The dielectric constant of aqueous solutions can be lowered by adding water-soluble organic
solvents, such as ethanol or acetone.
• The amount of organic solvent required to cause precipitation depends on the protein and
therefore proteins can be separated on this basis.
• The optimum quantity of organic solvent required to precipitate a protein varies from about 5 to
60%. Solvent fractionation is usually performed at 0 oC or below to prevent protein denaturation
caused by temperature increases that occur when organic solvents are mixed with water.
Denaturation of Contaminating Proteins
• Many proteins are denatured and precipitate from solution when heated above
a certain temperature or by adjusting a solution to highly acid or basic pHs.
Proteins that are stable at high temperature or at extremes of pH are most
easily separated by this technique because contaminating proteins can be
precipitated while the protein of interest remains in solution.
SEPARATION DUE TO DIFFERENT
ADSORPTION CHARACTERISTICS
Separation due to Different Adsorption
Characteristics
• Adsorption chromatography involves the separation of compounds by selective adsorption-
desorption at a solid matrix that is contained within a column through which the mixture
passes. Separation is based on the different affinities of different proteins for the solid matrix.
Affinity and ion-exchange chromatography are the two major types of adsorption
chromatography commonly used for the separation of proteins. Separation can be carried out
using either an open column or high-pressure liquid chromatography.
Ion Exchange Chromatography
• Ion exchange chromatography relies on the reversible adsorption-desorption
of ions in solution to a charged solid matrix or polymer network.
• This technique is the most commonly used chromatographic technique for
protein separation.
• A positively charged matrix is called an anion-exchanger because it binds
negatively charged ions (anions).
• A negatively charged matrix is called a cation-exchanger because it binds
positively charged ions (cations).
• The buffer conditions (pH and ionic strength) are adjusted to favor maximum
binding of the protein of interest to the ion-exchange column.
• Contaminating proteins bind less strongly and therefore pass more rapidly
through the column. The protein of interest is then eluted using another buffer
solution which favors its desorption from the column (e.g., different pH or ionic
Affinity Chromatography
• Affinity chromatography uses a stationary phase that consists of a ligand
covalently bound to a solid support. The ligand is a molecule that has a highly
specific and unique reversible affinity for a particular protein. The sample to be
analyzed is passed through the column and the protein of interest binds to the
ligand, whereas the contaminating proteins pass directly through. The protein
of interest is then eluted using a buffer solution which favors its desorption
from the column. This technique is the most efficient means of separating an
individual protein from a mixture of proteins, but it is the most expensive,
because of the need to have columns with specific ligands bound to them.
• Both ion-exchange and affinity chromatography are commonly used to
separate proteins and amino-acids in the laboratory. They are used less
commonly for commercial separations because they are not suitable for
rapidly separating large volumes and are relatively expensive.
SEPARATION DUE TO SIZE
DIFFERENCES
Separation Due to Size Differences

• Proteins can also be separated according to their size. Typically,

the molecular weights of proteins vary from about 10,000 to
1,000,000 daltons. In practice, separation depends on the
Stokes radius of a protein, rather than directly on its molecular
weight. The Stokes radius is the average radius that a protein
has in solution, and depends on its three dimensional molecular
structure. For proteins with the same molecular weight the
Stokes radius increases in the following order: compact globular
protein < flexible random-coil < rod-like protein.
Dialysis

• Dialysis is used to separate molecules in solution by use of semipermeable

membranes that permit the passage of molecules smaller than a certain
size through, but prevent the passing of larger molecules.
• A protein solution is placed in dialysis tubing which is sealed and placed
into a large volume of water or buffer which is slowly stirred.
• Low molecular weight solutes flow through the bag, but the large molecular
weight protein molecules remain in the bag.
• Dialysis is a relatively slow method, taking up to 12 hours to be completed.
It is therefore most frequently used in the laboratory.
• Dialysis is often used to remove salt from protein solutions after they have
Ultrafiltration

• A solution of protein is placed in a cell containing a semipermeable

membrane, and pressure is applied. Smaller molecules pass through the
membrane, whereas the larger molecules remain in the solution. The
separation principle of this technique is therefore similar to dialysis, but
because pressure is applied separation is much quicker. Semipermeable
membranes with cutoff points between about 500 to 300,000 are available.
That portion of the solution which is retained by the cell (large molecules) is
called the retentate, whilst that part which passes through the membrane
(small molecules) forms part of the ultrafiltrate. Ultrafiltration can be used to
concentrate a protein solution, remove salts, exchange buffers or
fractionate proteins on the basis of their size. Ultrafiltration units are used in
the laboratory and on a commercial scale.
Size Exclusion Chromatography
• This technique, sometimes known as gel filtration, also separates proteins
according to their size. A protein solution is poured into a column which is
packed with porous beads made of a cross-linked polymeric material (such as
dextran or agarose). Molecules larger than the pores in the beads are
excluded, and move quickly through the column, whereas the movement of
molecules which enter the pores is retarded. Thus molecules are eluted off the
column in order of decreasing size. Beads of different average pore size are
available for separating proteins of different molecular weights. Manufacturers
of these beads provide information about the molecular weight range that they
are most suitable for separating. Molecular weights of unknown proteins can
be determined by comparing their elution volumes Vo, with those determined
using proteins of known molecular weight: a plot of elution volume versus
log(molecular weight) should give a straight line. One problem with this
method is that the molecular weight is not directly related to the Stokes radius
SEPARATION BY
ELECTROPHORESIS
Separation by Electrophoresis
• Electrophoresis relies on differences in the
migration of charged molecules in a solution when
an electrical field is applied across it. It can be used
to separate proteins on the basis of their size, shape
or charge.
Non-denaturing Electrophoresis
• In non-denaturing electrophoresis, a buffered solution of native
proteins is poured onto a porous gel (usually polyacrylamide,
starch or agarose) and a voltage is applied across the gel. The
proteins move through the gel in a direction that depends on the
sign of their charge, and at a rate that depends on the magnitude
of the charge, and the friction to their movement:
• Proteins may be positively or negatively charged in solution depending on their isoelectic points (pI) and
the pH of the solution. A protein is negatively charged if the pH is above the pI, and positively charged if
the pH is below the pI.
• The magnitude of the charge and applied voltage will determine how far proteins migrate in a certain
time. The higher the voltage or the greater the charge on the protein the further it will move.
• The friction of a molecule is a measure of its resistance to movement through the gel and is largely
determined by the relationship between the effective size of the molecule, and the size of the pores in
the gel.
• The smaller the size of the molecule, or the larger the size of the pores in the gel, the lower the
resistance and therefore the faster a molecule moves through the gel.
• Gels with different porosity's can be purchased from chemical suppliers, or made up in the laboratory.
Smaller pores sizes are obtained by using a higher concentration of cross-linking reagent to form the
gel.
• Gels may be contained between two parallel plates, or in cylindrical tubes. In non-denaturing
electrophoresis the native proteins are separated based on a combination of their charge, size and
shape.
Denaturing Electrophoresis
• In denaturing electrophoresis proteins are separated primarily on their molecular weight.
• Proteins are denatured prior to analysis by mixing them with mercaptoethanol, which
breaks down disulfide bonds, and sodium dodecyl sulfate (SDS), which is an anionic
surfactant that hydrophobically binds to protein molecules and causes them to unfold
because of the repulsion between negatively charged surfactant head-groups.
• Each protein molecule binds approximately the same amount of SDS per unit length.
Hence, the charge per unit length and the molecular conformation is approximately
similar for all proteins.
• As proteins travel through a gel network they are primarily separated on the basis of
their molecular weight because their movement depends on the size of the protein
molecule relative to the size of the pores in the gel: smaller proteins moving more rapidly
through the matrix than larger molecules. This type of electrophoresis is commonly
called sodium dodecyl sulfate -polyacrylamide gel electrophoresis, or SDS-PAGE.
• To determine how far proteins have moved a tracking dye is added to the
protein solution, e.g., bromophenol blue. This dye is a small charged
molecule that migrates ahead of the proteins. After the electrophoresis is
completed the proteins are made visible by treating the gel with a protein
dye such as Coomassie Brilliant Blue or silver stain. The relative mobility of
each protein band is calculated:
 Electrophoresis is often used to determine the protein composition of food products. The protein is
extracted from the food into solution, which is then separated using electrophoresis. SDS-PAGE is used to
determine the molecular weight of a protein by measuring Rm, and then comparing it with a calibration
curve produced using proteins of known molecular weight: a plot of log (molecular weight) against relative
mobility is usually linear. Denaturing electrophoresis is more useful for determining molecular weights than
non-denaturing electrophoresis, because the friction to movement does not depend on the shape or
original charge of the protein molecules.
Isoelectric Focusing Electrophoresis

• This technique is a modification of electrophoresis, in which proteins are separated by charge on a

gel matrix which has a pH gradient across it. Proteins migrate to the location where the pH equals
their isoelectric point and then stop moving because they are no longer charged. This methods
has one of the highest resolutions of all techniques used to separate proteins. Gels are available
that cover a narrow pH range (2-3 units) or a broad pH range (3-10 units) and one should
therefore select a gel which is most suitable for the proteins being separated.
• Two Dimensional Electrophoresis
• Isoelectric focusing and SDS-PAGE can be used together to improve resolution of complex
protein mixtures. Proteins are separated in one direction on the basis of charge using isoelectric
focusing, and then in a perpendicular direction on the basis of size using SDS-PAGE.
• Amino Acid Analysis
• Amino acid analysis is used to determine the amino acid composition of
proteins. A protein sample is first hydrolyzed (e.g. using a strong acid) to
release the amino acids, which are then separated using chromatography,
e.g., ion exchange, affinity or absorption chromatography.