2. The technique of paper electrophoresis is
simple and inexpensive and requires only
micro quantities of plasma for separation.
The support medium is a filter paper
The electrophoresis apparatus in its simplest
form consists of two troughs to contain buffer
solution, through which electric current is
Frequently used in isolating proteins, amino
acids and oligopeptides.
“The charge carried by a molecule depends on the pH
of the medium. Electrophoresis at low voltage is not
usually to separate low molecular weight compounds
because of diffusion, but it is easier to illustrate the
relationship between charge and pH with amino
acids than with proteins (or) other macromolecules”.
4. The equipment required for electrophoresis consist
s basically of two items, a POWER PACK and an
Power pack :provides a stabilized direct current
and has controls for both voltage & current output,
which have an output of 0 to 500V and 0 to 150mA
The Electrophoretic cell: contains
the electrodes, buffer reservoirs, a support for
paper and a supporting transparent insulating cover.
The electrodes are usually made of platinum.
5. The two arrangements of the filter strips are commonly
used. The horizontal & vertical arrangements. Both the
arrangements are equally viable & the choice usually depends
upon personal preferences.
Paper of good quality should contain at least 95% α-cellulose
and should have only a very slight adsorption
7. 1) A long strip of filter paper is moistened with a
suitable buffer solution of the desired p H 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.
4)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.
8. 5)Homogeneous group of particles migrate as a
6)The electrophoresis is carried out for 16-18 hours.
7) Separated Proteins are fixed to a solid support
using a fixative such as Acetone or Methanol
8)Proteins are stained (bromophenol blue) to make
9) The separated proteins appear as distinct bands
10)Drawback-long time interval and blurring of
9. The different fractions appear as blue colored bands
across the filter paper starting from the moving
If a quantitative estimation is required for each
fraction, the bands may be carefully cut and eluted,
or the bands may be scanned optically in a
In human plasma five different bands can be
identified on paper electrophoresis
10. Serum analysis for diagnostic purpose is routinely
carried about by paper electrophoresis.
Muscle proteins, egg white proteins, milk proteins &
snake, insect venom analysis done by this technique.
12. Gel electrophoresis is a process that separates
fragments of DNA based on their sizes by using
electricity to carry them through a gel.
Separation is brought about through molecular
sieving technique, based on the molecular size of the
substances. Gel material acts as a "molecular sieve”.
Gel is a colloid in a solid form (99% is water).
It is important that the support media is electrically
13. The larger the fragments whether DNA,RNA or
protien,the slower it will move through the gel
DNA can be separated by fragment size, smallest
fragment traveling largest distance and larger
fragment traveling shortest distance.
Different types of gels which can be used are; Agar
and Agarose gel, Starch, Sephadex, Polyacrylamide
14. Agar gel is used for separation of different types of
protein mixtures as well as nucleic acids.
Polyacrylamide is most suitable for separation of
It is also frequently used in separating proteins,
peptides and amino acids from microgram quantities
of mixed samples.
15. Any charged ion or molecule migrates when placed
in an electric field, the rate of migration depend
upon its net charge, size, shape and the applied
16. Step1.Place DNA into tubes :DNA can come from
tissue or body fluid, such as cheek cells, blood, skin,
Step 2.Polymerase Chain Reaction: The polymerase
chain reaction uses a machine called a thermo cycler
to quickly copy a piece of DNA. Continue these three
steps 30 to 40 times to get lots of copies of DNA.
Step 3.Place restriction enzymes into DNA:The
restriction enzymes cut the DNA into different sizes
according to it’s sequence.
Step 4.Dye DNA and place into gel : The gel is made
out of wells at one end so that DNA can be loaded
into the gel.
17. Step 5 Run electric current through gel: DNA is
negatively charged so it will move towards the
positively charged end of the gel.
Step 6 Smaller pieces of DNA travel farther than
Larger pieces of DNA
22. Polyacrylamide gel electrophoresis (PAGE), describes a
technique widely used
in biochemistry,forensics,genetics,molecular biology and
Used to separate biological marocmolecules,usually,protiens or
nucleic acid according to their electrophoretic mobility.
Electrophoretic mobility is a function of the length,
conformation and charge of the molecule.
Polyacrylamide gel electrophoresis is a powerful tool used to
analyze RNA samples.
When polyacrylamide gel is denatured after electrophoresis, it
provides information on the sample composition of the RNA
23. Structure of acryl amide (CH2=CH-CO-NH2)
Polyacrylamide gel structure held together by
24. Polyacrylamide gels are tougher than agarose gels
It is thermo stable, transparent, strong and relatively
Proteins are separated on the basis of charge to
mass ratio and molecular size, a phenomenon called
Gels are made by free racially induced
polymerization of acrylamide and N,N’-
It is the most widely used technique.
26. Used for estimation of molecular weight of proteins and
Determination of subunit structure of proteins.
Purification of isolated proteins.
Monitoring changes of protein content in body fluids.
To identify whether a particular protein is pure or not.
Separation of proteins, prior to Western Blot transfer.
To measure genetic diversity
APPLICATIONS OF PAGE
29. SDS-PAGE (sodium dodecyl sulfate–polyacrylamide
Modified version of PAGE
Also called Denatured PAGE
It is a variant of polyacrylamide gel electrophoresis,
an analytical method in biochemistry for the
separation of charged molecules in mixtures by their
molecular masses in an electric field.
It uses sodium dodecyl sulfate (SDS) molecules to
help identify and isolate protein molecules.
30. It denatures proteins by binding to the protein chain
with its hydrocarbon ‘tail’, exposing normally buried
regions and ‘coating’ the protein chain with
The polar ‘head’ group of SDS adds an additional
benefit to the use of this denaturant.
SDS-PAGE is an electrophoresis method that allows
protein separation by mass.
The medium (also referred to as ′matrix′) is a
polyacrylamide-based discontinuous gel.
31. SDS acts as surfactants, covering the proteins'
intrinsic charge and conferring them very similar
The intrinsic charges of the proteins are negligible
in comparison to the SDS loading, and the positive
charges are also greatly reduced in the basic pH
range of a separating gel.
Upon application of a constant electric field, the
protein migrate towards the anode, each with a
different speed, depending on its mass.
This simple procedure allows precise protein
separation by mass.
34. In their native form, proteins fold into a variety of shapes,
some compact, some elongated.
The rate of migration of native proteins through a sieving
medium is therefore more a reflection of their relative
compactness, and less an accurate measure of molecular
Denaturing the proteins nullifies structural effects on
mobility, allowing separation on a true charge/mass ratio
It also separates subunits in multimeric proteins, allowing
analysis of large, complex aggregates.
WHY SDS ?
36. SDS is a anionic detergent (soap) that can dissolve
hydrophobic molecules but also has a negative
For uniform distribution of charge per unit
For getting the uniform direction of motion of
If a cell is incubated with SDS, the membranes will
be dissolved and the proteins will be solubilized by
SIGNIFICANCE OF SDS
38. Also called as Native PAGE
It is an electrophoresis method to separate native
The conditions are set such that the migrating
proteins are kept in their native state. The buffers
provide a non-denaturing, and the electrophoresis is
performed at low temperature in order to dissipate
Many enzymes retain their native conformation and
their enzymatic activities while running in the gel.
39. If certain conditions apply, these enzymes can be highly
selectively detected within the gel through a specific
‘staining’ reaction even in the presence of a large excess
of ‘contaminating’ proteins.
After completion of electrophoresis, the gel is soaked in a
solution containing the substrate of the enzyme.
But most enzymes do not have such natural substrates.
However, once the molecular mechanism of catalysis is
revealed, synthetic substrates can be designed that, on
the one hand, mimic natural substrates and, on the other
hand, lead to colorful insoluble products.
40. As the substrate is usually a small molecule, it
quickly diffuses into the gel while the large enzyme
molecules do not diffuse out.
In an optimal case, the natural product of the
enzymatic reaction is a colored and insoluble
compound that precipitates inside the gel and marks
the exact location of the enzyme.
41. Useful method for checking the uniformity of the isolated
Even if the purified protein sample contains only a single type
of protein, the sample might not be uniform. Some of the
molecules might be unfolded or have undergone chemical
modifications. It changes the overall shape of the molecule,
and chemical modifications change the electric charge of
native molecules. These alterations can be detected after
traditional staining of the purified sample. If no such side
products are present, protein molecules will run in a single
sharp band. Otherwise, multiple bands or smearing of the
band is expected.
42. Used to detect complex formation between proteins.
If two (or more) proteins form a complex, the complex can be
detected as an extra band in the gel. This is because in native-
like conditions, many non-covalent interactions are
maintained and the complex migrates apparently as a single
It is highly important to pay attention to the relationship of the pI values of the
proteins or protein complexes and the pH of the gel buffer, as this will determine
where individual proteins will migrate in the gel.
44. 4/1/2018 44
Native PAGE SDS PAGE
Separation is based upon charge, size,
and shape of macromolecules.
Separation is based upon the molecular
weight of proteins.
Useful for separation and/or purification
of mixture of proteins
The most common method for
determining MW of proteins
This was the original mode of
Very useful for checking purity of protein