UNIT IV

BIOANALYTICAL TECHNIQUES

Electrophoresis – Principle, procedure and applications of electrophoresis (paper

electrophoresis, gel electrophoresis – PAGE, SDS- PAGE and agarose gel electrophoresis) and
isoelectric focusing.
ELECTROPHORESIS

Electrophoresis and its principle

Electrophoresis may be defined as the migration of the charged particle through a solution
under the influence of an external electrical field.

Ions that are suspended between two electrodes tend to travel towards the electrodes that
bears opposite charges. Depending on kind of charge the molecule carry, they move towards either:
To cathode or to Anode. An ampholyte become positively charged in acidic condition and migrate
to cathode, in alkaline condition they become negatively charge and migrate to anode.

The rate of migration of an ion in electrical field depend on factors:

1. Net charge of molecule
2. Size and shape of particle
3. Strength of electrical field
4. Properties of supporting medium
5. Temperature of operation

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Types of electrophoresis:

It is a type of protein separation method which relies on protein sizes to segregate the
mixture. It is one of the highly efficient techniques of analysis and sole method for separation of
proteins for western blot, RNA studies etc. But, on negative side it also time-consuming, expensive
and technical skilled procedure due to which is less preferred in health care. Electrophoresis is
similar to other separation techniques like chromatography but it differs in-terms of the types of
samples analyzed, the method used for separation, principle used etc.

FREE ELECTROPHORESIS
In this type of electrophoresis a free electrolyte is taken in place of supporting media. It is
mostly of two types-
i.Micro Electrophoresis : It is mostly used in calculating Zeta potentials(a colloidal property of
cells in a liquid medium)of the cells.
ii.Moving boundary Electrophoresis which for many years had been used for quantitative analysis
of complex mixtures of macromolecules esp. Proteins.

ZONE ELECTROPHOROSIS
It involves the migration of the charged particle on the supporting media can be Paper,
Cellulose acetate membrane, Starch Gel, Polyacrylamide. Components separated are distributed
into discrete zone on the support media. Supporting media is saturated with buffer solution, small
volume of the sample is applied as narrow band.
Advantages: Useful in biochemical investigations. Small quantity of sample can be analysed. Cost
is low and easy maintenance.
Disadvantages: Unsuitable for accurate mobility and isoelectric point determination. Due to the
presence of supporting medium, there are technical complications.

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Different types of zone electrophoresis:
 Paper electrophoresis
 Cellulose acetate electrophoresis
 Gel electrophoresis

PAPER ELECTROPHOROSIS

Principle:
Paper Electrophoresis is one of the types of zone electrophoresis. When charged molecules are
placed in an electric field, they migrate toward either the positive or negative pole according to their
charge. In contrast to proteins, which can have either a net positive or net negative charge, nucleic
acids have a consistent negative charge imparted by their phosphate backbone, and migrate towards
the anode.
Electrophoretic unit is of two types:
1. Horizontal electrophoretic unit
2. Vertical electrophoretic unit

Fig: Horizontal Paper electrophoresis

Fig: Vertical Paper electrophoresis

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Equipments:
The equipment required for electrophoresis consists basically of two items, a power pack and
electrophoretic cell.
1. Power pack: Power pack provides a stabilized direct current and has controls for both voltage
and current out put.
2. The Electrophoretic cell: It contains: the electrodes, buffer reservoirs, a support for paper and a
transparent insulating cover. The electrodes are usually made of platinum.

Working:
1) A long strip of filter paper is moistened with a suitable buffer solution of the desired pH and
the sample is applied transversely across the central part of the strip.
2) Ends are fixed to dip in buffer solutions in two troughs fitted with electrodes.
3) Electric field of about 20 volts/cm is established. The charged particles of sample migrate
along the strip towards respective electrodes of opposite polarity, according to net charges,
sizes and interactions with the solid matrix.
4) Homogeneous group of particles migrate as a separate band.
5) The electrophoresis is carried out for 16-18 hours.
6) Proteins are stained (bromophenol blue) to make them visible.
7) The separated proteins appear as distinct bands.

Applications:
1) Paper electrophoresis has emerged as a simple, inexpensive and accurate laboratory
procedure for various research and clinical studies.
2) Clinical applications of paper electrophoresis include study of sickle cell disease,
haemoglobin abnormalities, and separation of blood clotting factors and serum plasma
proteins from blood sample.
3) Used in separation and identification of alkaloids.
4) Used for testing water samples, toxicity of water, and other environmental components.
5) Drug-testing industry uses paper electrophoresis to determine presence of illegal drugs
crime suspects.
6) Used by the investigators and in forensics to analyze inks used in currency to check the
counterfeiters.

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 CELLULOSE ACETATE ELECTROPHORESIS

Kohn in 1958 introduced, Cellulose acetate as a medium for electrophoresis. It was

developed from bacteriological cellulose acetate membrane filters and is commercially available
as high purity cellulose acetate strips, which are thin and have a uniform micropore structure.
Buffers used in both the electrophoresis i.e., in paper and cellulose acetate electrophoresis
are same.
 It has the advantage over the paper in that it is a much more homogeneous medium, with
uniform pore size and does not absorb proteins like the paper.
 Far simpler to set up and run, single samples are normally run on cellulose acetate strips
although multiple samples are frequently run on wider sheets.
 The cellulose acetate is first method in electrophoresis buffer (pH 8.6 for serum) and 1 to 2 l
sample is loaded.
 The end of the strip makes contact with the electrophoresis buffer tanks via a filter paper
wick and electrophoresis is conducted at 6-8 V/cm for about 3hrs.
 Following electrophoresis, the strip is stained for protein, destained, and the band is
visualized.

Applications:
 Cellulose acetate is especially used for clinical investigations such as separation of
hemoglobin from blood, lipoproteins and glycoproteins.
 One of the older methods, and has a number of applications particularly used in the
clinical analysis of serum samples.
 Alternative to paper electrophoresis.

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 GEL ELECTROPHORESIS

Gel electrophoresis is the most common method to carry out the process of electrophoresis.
It makes use of gel as a support matrix. It is the most popular and commonly used method. Used for
both analytical and preparative processes.
Separation is brought about through molecular sieving technique, based on the molecular
size of the substances. Gel material acts as a “molecular sieve”. It is important that the support
media is electrically neutral.
Different types of gels which can be used are; Agar and Agarose gel, Starch, Sephadex,
Polyacrylamide gels. A porous gel acts as a sieve by retarding or, in some cases, by completely
obstructing the movement of macromolecules while allowing smaller molecules to migrate freely.

Principle:
In this, porous gel matrix is used which consist of the cross-linked polymer network.
Through this network, molecules of different size, charge and shape pass through. This relies upon
the fact that negatively charged molecule will attract towards the positive end and vice versa. After
the migration, bands will appear on the gel matrix at different levels. Those which lag behind will
be the heavy molecules and those which moves faster are lighter molecules through the pores of the
gel matrix.

Types of Gel electrophoresis

I. Vertical (PAGE, SDS-PAGE)
II. Horizontal (Agarose gel electrophoresis)

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 I. Vertical (PAGE, SDS-PAGE)

POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)

Principle:
Electrophoresis of protein in polyacrylamide gel is advantageous than in any other
gel and are carried out in buffers gels (poly acrylamide native gels). The presence of
isoenzyme is analysed in Native gel electrophoresis. Polyacrylamide gels are formed by
polymerizing acrylamide with a cross-linker (bisacrylamide) in the presence of a catalyst
(sulphate radical) and chain initiator tetramethylethylene diamine (TEMED). The porocity of
the gel is determined by the relative proportion of acrylamide monomer to bisacrylamide. Gels are
usually referred in terms of total percentage of acrylamide and bis present, and most protein
separations are performed using gels in the range 7-15%. A low percentage of the gel is
used to separate the high molecular weight proteins and vice -versa.

Instrumentation:

The electrophoresis apparatus is set up with cathode buffer covering the gel in the
negative electrode chamber, and anode buffer in the lower positive electrode chamber. Next, the
denatured sample proteins are added to the wells to one end of the gel with a micropipette. Finally,
the apparatus is hooked up to a power source under appropriate running conditions to
separate the protein bands.
An electric field is applied across the gel, causing the negatively-charged proteins to
migrate across the gel towards the positive (+) electrode (anode). Depending on their size, each
protein will move differently through the gel matrix: short proteins will more easily fit through the
pores in the gel, while larger ones will have more difficulty (they encounter more
resistance).
After a set amount of time (usually a few hours), the proteins will have differentially
migrated based on their size; smaller proteins will have travelled farther down the gel, while

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larger ones will have remained closer to the point of origin. Therefore, proteins may be
separated roughly according to size (and therefore, molecular weight).

Procedure:
1. Equipment - Glass plates, spacers, a comb (used to create the sample wells) and
casting frame are required to make the gel.
2. Buffers - Tris-based buffers are used for PAGE. Three categories of buffer are
necessary:
 Gel casting buffer (used to make the gel)
 Sample buffer
 Running buffer (fills the gel tank where electrophoresis takes place)
3. The gel - A protein gel is formed in two sections, the stacking gel and the resolving
gel. The role of the stacking gel is to allow sample loading and to guide the samples
into the top of the resolving gel, so they all enter at the same time. The proteins
within the sample will then be separated so they can be “resolved” in the resolving
gel. The optimal gel percentage will depend upon the sizes of the proteins to be
separated.
4. Sample preparation - For native PAGE, SDS and β-mercaptoethanol are not
included in the loading dye and no heating step is performed to maintain the proteins
in their native conformation.

5. Controls- Size markers are normally incorporated on either end of a sample row to
enable size estimates for any bands detected.
6. Running the gel - Part-fill the electrophoresis tank with an appropriate running
buffer, often TBE for native PAGE. The voltage used and run time will vary
depending on the percentage resolving gel being used.
7. Staining and visualization - Once the samples have migrated a sufficient distance
down the gel, the gel is carefully removed from the glass plates. The proteins are
stained using Coomassie brilliant blue and visualized.
After staining, different species biomolecules appear as distinct bands within
the gel. It is common to run molecular weight (size) markers of known molecular
weight in a separate lane in the gel to calibrate the gel and determine the
approximate molecular mass of unknown biomolecules by comparing the distance
travelled relative to the marker.

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 Applications of Polyacrylamide Gel Electrophoresis (PAGE)
 Measuring molecular weight.
 Peptide mapping.
 Estimation of protein size.
 Determination of protein subunits or aggregation structures.
 Estimation of protein purity.
 Protein quantitation.
 Monitoring protein integrity.
 Comparison of the polypeptide composition of different samples.
 Analysis of the number and size of polypeptide subunits.
 Post-electrophoresis applications, such as Western blotting.
 Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic
Acid.
 Pouring and Running a Protein Gel by reusing Commercial Cassettes.
 Detection of Protein Ubiquitination.

Advantages

 Stable, chemically cross-linked gel that is stable and stable.

 Greater resolution capacity (Sharp bands)

 can accommodate greater quantities of DNA with no significant loss in resolution

 The DNA that is extracted from polyacrylamide gels are extremely pure

 The pores of polyacrylamide gels is adjustable in a simple and manageable manner by

altering the concentrations of two monomers.

 It is useful for separating smaller molecular weight pieces.

Disadvantages

 The process is typically more difficult to handle and prepare taking longer to prepare.

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 SODIUM DODECYL SULPHATE - POLYACRYLAMIDE GEL
ELECTROPHORESIS (SDS-PAGE)

Sodium Dodecyl Sulphate (SDS) polyacrylamide gel electrophoresis is mostly used to

separate proteins accordingly by size. This is one of the most powerful techniques to separate
proteins on the basis of their molecular weight.

Principle:
This technique uses anionic detergent SDS which dissociates proteins into their individual
polypeptide subunits and gives a uniform negative charge along each denatured polypeptide.
SDS also performs another important task. It forces polypeptides to extend their conformations to
achieve similar charge : mass ratio. The rate of movement is influenced by the pore size of the gel
and the strength of electric field. In SDS-PAGE the vertical gel apparatus is mostly used. Although
it is used to separate proteins on a routine basis, SDS-PAGE can also be used to separate DNA and
RNA molecules.
The molecular weight of protein may be estimated if they are subjected to electrophoresis in
the presence of SDS and a reducing agent mercaptoethanol. SDS disrupts the secondary, tertiary
and quaternary structure of the protein to produce a linear polypeptide chain coated with negatively
charged SDS molecules. Mercaptoethanol assists the protein denaturation by reducing all disulfide
bonds.

Procedure:

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 1) Sample preparation
 Samples may be any material containing proteins or nucleic acids.
 The sample to analyze is optionally mixed with SDS for proteins or urea for nucleic acids.
 A tracking dye (bromophenol blue) may be added to the solution.
 The sample is heated in boiling water bath for 2-3 min and cooled.

2) Preparation of polyacrylamide gel

 The gels typically consist of acrylamide, bisacrylamide, the denaturant (SDS or urea), chain
initiator tetra methyl ethylene diamine (TEMED) Ammonium per sulphate (APS) which helps
in polymerization and a buffer with an adjusted pH.
 Gels are usually polymerized between two glass plates in a gel caster, with a comb inserted at
the top to create the sample wells.
 After the gel is polymerized the comb can be removed and the gel is ready for
electrophoresis.

3) Electrophoresis
 Various buffer systems are used in PAGE depending on the nature of the sample and the
experimental objective.
 The sample is injected into the wells.
 An electric field is applied across the gel, causing the negatively charged proteins or nucleic
acids to migrate across the gel away from the negative and towards the positive electrode (the
anode).
 Current of 10-15 mA is applied until the samples travel through the stacking gel. Then run at
30 mA until the bromophenol blue reaches the bottom of the gel. The proteins within the
sample will then be separated so they can be “resolved” in the resolving gel.

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  Depending on their size, each biomolecule moves differently through the gel matrix. The gel
is run usually for a few hours.
4) Detection
 Following electrophoresis, the gel may be stained with Coomassie Brilliant Blue or
autoradiography.
 After staining, different species biomolecules appear as distinct bands within the gel.

Advantages:

1. Mobility of the molecules is high and separation is rapid.

2. All the proteins are negatively charged; therefore, all migrate towards anode.

3. The proteins treated with SDS fixed dyes are better than the native proteins.

4. SDS solubilizes all proteins, including very hydrophobic and even denatured proteins.

Applications:

SDS-PAGE has many applications. It is mostly used for following purposes:

1. Establishing protein size

2. Protein identification

3. Determining sample purity

4. Identifying disulfide bonds

5. Quantifying proteins

6. Blotting applications

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 II. Horizontal (Agarose gel electrophoresis)

AGAROSE GEL ELECTROPHORESIS

Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry,

molecular biology, genetics, and clinical chemistry to separate a mixed population of
macromolecules such as DNA, RNA or proteins in a matrix of agarose.

Principle:
Agarose is a natural linear polymer extracted from seaweed that forms a gel matrix by
hydrogen-bonding when heated in a buffer and allowed to cool. They are the most popular medium
for the separation of moderate and large-sized nucleic acids and have a wide range of separation.
The agarose gel consists of microscopic pores that act as a molecular sieve that separates molecules
based on the charge, size, and shape.
When a potential difference is applied across the electrodes of a horizontal electrophoretic
tank containing agarose gel and biomolecules (such as nucleic acids) are loaded, then they get
separated according to their molecular size (bigger molecules have more molecular size and smaller
molecules have small molecular size) and move to their respective electrodes. Here, the agarose gel
acts as a sieve. The larger and the bulky molecules stay behind whereas the smaller molecules move
faster and quickly towards their respective electrodes. The DNA will be visualized by including in
the gel an intercalating dye, ethidium bromide.

Instrumentation:
The equipment and supplies necessary for conducting agarose gel electrophoresis are
relatively simple and include:
1. An electrophoresis chamber and power supply.
2. Gel casting trays, which are available in a variety.
3. Sample combs, around which molten medium is poured to form sample wells in the gel.
4. Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
5. Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to
“fall” into the sample wells, and one or two tracking dyes, which migrate in the gel and
allow visual monitoring or how far the electrophoresis has proceeded.
6. Staining: DNA molecules are easily visualized under an ultraviolet lamp when
electrphoresed in the presence of the extrinsic fluor ethidium bromide.
7. Transilluminator which is used to visualize stained DNA in gels.

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Procedure:

1. To prepare gel, agarose powder is mixed with electrophoresis buffer to the desired
concentration, and heated in a microwave oven to melt it.
The concentration of Agarose Gel:
 The percentage of agarose used depends on the size of fragments to be resolved.
 if the aim is to separate large DNA fragments, a low concentration of agarose should be
used, and if the aim is to separate small DNA fragments, a high concentration of agarose is
recommended.
2. Ethidium bromide is added to the gel to facilitate visualization of DNA after electrophoresis.
3. After cooling the solution to about 60oC, it is poured into a casting tray containing a sample
comb and allowed to solidify at room temperature.
4. After the gel has solidified, the comb is removed, taking care not to rip the bottom of the
wells.
5. The gel, still in plastic tray, is placed horizontally into the electrophoresis chamber and is
covered with buffer.
6. Samples containing DNA mixed with loading buffer are then pipetted into the sample wells,
the lid and power leads are placed on the apparatus, and a current is applied.
7. DNA will migrate towards the positive electrode ie, anode.
8. The distance DNA has migrated in the gel can be judged by visually monitoring migration
of the tracking dyes like bromophenol blue and xylene cyanol dyes.

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Application of Agarose Gel Electrophoresis:

1. Separation of restriction enzyme digested DNA including genomic DNA, prior to Southern Blot
transfer.
2. It is often used for separating RNA prior to Northern transfer.
3. Analysis of PCR products after polymerase chain reaction to assess for target DNA
amplification.
4. Allowing estimation of the size of DNA molecules using a DNA marker or ladder which
contains DNA fragments of various known sizes.
5. Allows the rough estimation of DNA quantity and quality.
6. Quality of DNA is assessed by observing the absence of streaking or fragments (or
contaminating DNA bands).
7. Other techniques rely on agarose gel electrophoresis for DNA separation including DNA
fingerprinting.

Advantages and Disadvantages of Agarose Gel Electrophoresis:

• The advantages are that the gel is easily poured, and does not denature the samples. The
samples can also be recovered.
• The disadvantages are that gels can melt during electrophoresis, the buffer can become
exhausted, and different forms of genetic material may run in unpredictable forms.

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 ISOELECTRIC FOCUSING (IEF)

Isoelectric focusing (IEF), also known as electrofocusing, is a technique for

separating different molecules by differences in their isoelectric point (pI). The isoelectric
point (pI) is the pH at which a particular molecule carries no net electrical charge. It is a type
of zone electrophoresis usually performed on proteins in a gel that takes advantage of the fact
that overall charge on the molecule of interest is a function of the pH of its surroundings.

Principle:
The main principle of isoelectric focusing is that two different proteins having different
isoelectric points migrate in pH gradient and presence of electric field till total charge of a
protein gets to zero. The migration stops as the condition is achieved.
Protein gets immobilized in the respective pH gradient while approaching their pI. The
gel is then stained and results are documented. After the separation of protein 2D gel
electrophoresis is used for further separation of protein with respect to their mass.
When net charge of protein gets to zero but cannot be further moved in presence of an
electric field, protein remains in its position due to gradient force and this phenomenon is
known as “focusing”. Reduction in sample or increase in voltage will result in appearance of
more clear bands.
The minimum pI is used for estimating separation. The charge of protein is the sum
of its negative and positive charges. These charges are dependent on the pH of their
surroundings. The pI of a protein is low if the acidic group is more than the basic group. The pI
of a protein will be high if the basic group is more than the acidic group. Proteins get a
positive charge when the surrounding pH is below their pI. Protein gets a negative charge
when pH surrounding the protein is higher than the protein’s pI.

Procedure
IEF involves adding an ampholyte solution into immobilized pH gradient (IPG) gels.
IPGs are the acrylamide gel matrix co-polymerized with the pH gradient. A protein that is in a
pH region below its pI will be positively charged and so will migrate toward the cathode.
As it migrates through a gradient of increasing pH, the protein's overall charge will decrease
until the protein reaches the pH region that corresponds to its pI. At this point it has no net
charge and so migration stops. As a result, the proteins become focused into sharp stationary
bands.

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 The technique is capable of extremely high resolution with proteins differing by a
single charge being fractionated into separate bands.
Molecules to be focused are distributed over a medium that has a pH gradient (usually
created by aliphatic ampholytes). The ampholytes used are polyamino polycarboxylic acid. It
is available as Bio-Lyte and Pharmalyte commercially. Later an electric current is passed
through the medium.
Negatively charged molecules migrate through the pH gradient in the medium toward
the "positive" end while positively charged molecules move toward the "negative" end. As a
particle moves toward the pole opposite of its charge it moves through the changing pH
gradient until it reaches a point in which the pH of that molecules isoelectric point is reached.
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At this point the molecule no longer has a net electric charge and as such will not proceed any
further within the gel.
This technique is traditionally used as the first “dimension” of separation in a 2D-
PAGE experiment, in which molecules are first separated by their charge prior to further
separation by traditional SDS-PAGE, which is the second dimension of separation.

Advantages:
 Proteins that by as little as 0.001 pH units can be separated.
 As spreading of bands is minimized due to application of the applied field and the pH
gradient , high resolution can be achieved.
 Isoelectric focusing (IEF) is a powerful analytical tool for the separation of proteins.
 Performing IEF is easier because the placement of sample application is not important.

Disadvantages:
 Carrier ampholytes are generally used in high concentration, a high voltage (upto 2000v) is
necessary.
 Limited stability of solutions.

Applications
 For separating proteins and peptides.
 For research in Taxonomy, Cytology and Immunology etc.
 IEF gel is used as identity test when migration pattern on gel is compared with standard
preparation.
 It offers an effective alternative to conventional electrophoresis for genetic marker
typing.

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