SEPARATION PROCESSES IN BIOENGINEERING

Electrophoresis

Murat Topuzoğulları
 What is electrophoresis?
Electrophoresis refers to separation of charged solutes based
on their electrophoretic mobility i.e. movement of charged
molecules in response to an electric field.

Electrophoresis gives excellent solute separation and this

technique is extensively used for protein and nucleic acid
purification and analysis.

However, its use for preparative scale bioseparation is not yet

extensive.

Electrophoresis is the process of moving charged molecules in

solution by applying an electric field across the mixture.
 What is electrophoresis?

Molecules in an electric field move with a speed dependent

on their charge, shape, and size.

As an analytical tool, electrophoresis is simple and relatively

rapid.
Figure 1. Mechanism of moving the molecules through the gel according to their size.
 The velocity at which a charged molecule migrates in an electric field is given
by:

v = UEE
v = velocity (m/s)
UE = Electrophoretic mobility (m2/V s)
E = Strength of electric field (V/m)

ε: dielectric constant
z: zeta potential;
f(ka): Henry function
η: viscosity of the medium
 Which Molecules can be Analyzed
by electrophoresis

It is used chiefly for analysis and separation of

very large molecules such as proteins and nucleic
acids.
 But can also be applied to simpler charged
molecules, including charged sugars, amino acids,
peptides, nucleotides, and simple ions.
 Media or Matrix for electrophoresis
The most common stabilizing media used for electrophoresis
in research laboratories are
1. Polyacrylamide gel
2. Agarose gel
Proteins are separated by a discontinuous polyacrylamide
gel as a support medium and sodium dodecyl sulfate (SDS) to
denature the proteins.
Nucleic acids are separated on either polyacrylamide or
agarose gels.
Agarose and polyacrylamide gels are cross-linked, sponge-like
structures.
Although they are up to 99.5% water, the size of the pores of
these gels is similar to the sizes of many proteins and nucleic
acids.
As molecules are forced through the gel by the applied voltage,
larger molecules are retarded by the gel more than smaller
molecules.
Whichever matrix is selected, it is important that it be
electrically neutral.
 Agarose gels
Agarose is a highly purified polysaccharide derived from agar.
Agarose is normally purchased as a dry powder. It is dissolved
when the suspended powder is heated to near boiling and it
remains liquid until the temperature drops to about 40⁰C.
The pore size and sieving characteristics of a gel are
determined by adjusting the concentration of agarose in the
gel. The higher the concentration, the smaller the pore size.
Working concentrations are normally in the range of 0.4–4%
w/v.
 Agarose gels are relatively fragile and should be handled carefully.
The gels are hydrocolloids, held together by hydrogen and
hydrophobic bonds, and tend to be somewhat brittle.
1% gels are common for many applications.

A B C
Figure 2. Agarose gels. (A) In solid state no hydrogen and hydrophobic bonds, (B) In melting stage
formation of bonds, (C) Gel state form by noncovalent hydrogen and hydrophobic bonds between
long sugar polymers.
 Polyacrylamide gels
 Polyacrylamide (poly(2-propenamide) or poly(1-carbamoylethylene))

is a polymer(-CH2CHCONH2-) formed from acrylamide subunits. It is

highly water-absorbent, forming a soft gel when hydrated.

 Polyacrylamide gels are physically tougher than agarose gels.

 The gel forms when a mixed solution of acrylamide and cross-linker

monomers co-polymerize into long chains that are covalently cross-

linked. The most common cross-linker is N,N'-methylenebisacrylamide.

the average pore size of a gel is determined by the percentage of solids in
the gel .

Figure 3. Acrylamide gels have covalent cross-links between polymer strands.

 Special type of gel

When the method of electrophoresis first invented starch

gel was used as media.
This gel is partially hydrolyzed potato starch makes for
another non-toxic medium for protein electrophoresis.
It is slightly more opaque than acrylamide or agarose.
Non-denatured proteins can be separated according to
charge and size.
They are visualized using Napthol Black or Amido Black
staining. Typical starch gel concentrations are 5% to 10%.
 Protein Electrophoresis
Protein electrophoresis is a method for analyzing the
proteins in a fluid or an extract.
The electrophoresis may be performed with a small volume
of sample in a number of alternative ways with or without a
supporting medium: SDS polyacrylamide gel electrophoresis
or sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE).
 Each method has many variations with individual
advantages and limitations.
The most commonly used system is
also called the Laemmli method after
U.K. Laemmli, who was the first to
publish a paper employing SDS-PAGE in a
scientific study.

Figure 4. Band of proteins in

SDS-PAGE gel
Overall process of protein electrophoresis
 SDS (sodium dodecyl sulfate)
It is also called as lauryl sulfate.
SDS is an anionic detergent. Its molecules have a net
negative charge within a wide pH range.
This compound works by disrupting non-covalent
bonds in the proteins, denaturing them, and causing the
molecules to lose their native shape (from folding
structure to unfold into a rod-like shape) and form
negatively charged complexes.
The amount of SDS bound by a protein, and so the
charge on the complex, is roughly proportional to its
size.
Commonly, about 1.4 g SDS is bound per 1 g protein,
although there are exceptions to this rule.
SDS-polypeptide complexes migrated or moved
through the gels according to the size of the
polypeptide.
 Buffers and pH of proteins in SDS– PAGE

Proteins are amphoteric (or zwitterionic) compounds.

Therefore, they are charged either positively or negatively
because they contain both acidic and basic residues.
Electrophoretic protein separations are based on the
mobility of the different proteins, the pH of the solution must
be kept constant to maintain this charge
 Molecular-weight size marker
A molecular-weight size marker, also referred to as a protein
ladder or DNA ladder, is a set of standards that are used to
identify the approximate size of a molecule run on a gel
during electrophoresis.
Protein and DNA markers with pre-determined fragment sizes
and concentrations are commercially available. These can be run
in either agarose or polyacrylamide gels.
The markers are loaded in lanes adjacent to sample lanes
before the commencement of the run.
 DNA markers
Different DNA ladders are commercially available depending
on expected DNA length.
The 1kb ladder with fragment ranging from about 0.5 kbp to
10 or 12 kbp, and the 100 bp ladder with fragments ranging
from 100 bp to just above 1000 bp are frequently used.
There are also special DNA ladders for supercoiled DNA and
RNA.
Commercial DNA markers are usually supplied with the
loading dye mixture, due to the difficulty of visualizing DNA
during electrophoresis.
Figure 6. Different sized DNA marker or DNA ladder (on the basis of base pair)
Figure 7.Use of DNA marker to measure DNA banding of different samples
in Gel electrophoresis
 Protein markers
Protein markers can be pre-stained or unstained prior to loading.
It is of known molecular weight, that’s why the molecular
weight of the polypeptide chain(s) of protein can be estimated.
It is a mixture of 12 recombinant, highly purified proteins, which
resolve into clearly identifiable sharp bands from 10-250 kDa when
analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue.
Coomassie Brilliant Blue is the name of two
similar triphenylmethane dyes that were developed for use in the
textile industry but are now commonly used for staining proteins in
analytical biochemistry.
 Properties of Protein marker

Storage Temperature
 at -20°C

Storage Conditions
i. 70 mM Tris-HCl
ii. 33 mM NaCl
iii. 40 mM DTT
iv. 1 mM Na2EDTA
v. 10% Glycerol
vi. 0.01% bromophenol blue
vii. 2% SDS
viii.pH 6.8 at 25°C

Figure 8. SDS-PAGE band profile of protein marker

 A B

C
Figure 9. Processes of poring the medium and combing for electrophoresis.
Figure 10. Visual pore after solidifying the medium
Figure 11. Process of loading the DNA or protein sample .
 A B
Figure 12. Whole Electrophoresis system before starting the electricity flow on it;
(A) for DNA sample and (B) for protein sample.
Figure 13. After completing the process visual band are observer through the gel
Figure 14. Different bands under ultraviolet ray after electrophoresis
Figure 15 . Protein bands after staining
 Applications of Electrophoresis

Gel electrophoresis is used in molecular

biology, genetics, microbiology and biochemistry. The
results can be analyzed quantitatively by visualizing the gel
with UV light and a gel imaging device. The vast applications
of electrophoresis are most evident in the health or medical
industry, including antibiotic and vaccine analysis.
 DNA and RNA Analysis

Through DNA electrophoresis, specific DNA sequences

can be analyzed, isolated and cloned. It can be used in
forensic investigations and paternity tests.
Electrophoresis of RNA samples can be used to check for
genomic DNA contamination and also for RNA
degradation.
 Figure 16:DNA fingerprints of two children(C). Red matches
bands common with their mother(M);blue matches bands
common with their father (F)

Figure17. DNA profile of a child after

DNA electrophoresis
 Protein Analysis
Electrophoresis has advanced our understanding on the
structure and function of proteins.
This method is applicable for diagnosis processes. For
example - Diagnosis of human blood and urine samples.
Through electrophoresis, the amount of proteins in human
blood or urine is measured and compared to established
normal values. Lower or higher than the normal levels usually
indicates a disease.
 Antibiotics Analysis

 The application of electrophoresis in antibiotic studies

dates back to the 1950s specially for long spectrum
antibiotics .
 Such as penicillin, are among the widely prescribed drugs
against bacterial infections.
 With electrophoresis, experts are not only able to
synthesize new antibiotics but are also able to analyze
which types of bacteria are antibiotic-resistant.
 Vaccine Analysis
Vaccine analysis is one of the many important
applications of electrophoresis.
There are several vaccines that have been purified,
processed and analyzed through electrophoresis, such as
the influenza vaccine, hepatitis vaccine and polio vaccine.
 Proteomics
Proteomics has become another important application
area of electrophoresis. 2D electrophoresis is the most
frequently used technique.
In 2D electrophoresis, first dimension is isoelectric focus
(IEF) electrophoresis. Then, the IEF gel is loaded to SDS-
PAGE as the 2nd dimension.
This technique allows to visualize complex protein
mixtures, such as the changes in protein concentrations or
modifications in cells or tissues.
Proteomics