•Proteins are biochemical compounds consisting of one or
more polypeptides typically folded into a globular or fibrous
form, facilitating a biological function.
•Proteins are the most abundant biological macromolecules,
occurring in all cells and all parts of cells. Proteins also occur
in great variety; thousands of different kinds, ranging in size
from relatively small peptides to huge polymers with
molecular weights in the millions, may be found in a single
4. protein separation method
•SDS-PAGE (SDS-polyacrylamide gel
•High-performance liquid chromatography
•Thin-layer chromatography (TLC)
•Two-dimensional (2-D) gel electrophoresis
5. Thin-layer chromatography
Thin-layer chromatography (TLC) is
a chromatography technique used to separate non-
Thin-layer chromatography is performed on a sheet
of glass, plastic, or aluminium foil, which is coated
with a thin layer of adsorbent material, usually silica
gel, aluminium oxide (alumina), or cellulose. This
layer of adsorbent is known as the stationary phase.
After the sample has been applied on the plate,
a solvent or solvent mixture (known as the mobile
phase) is drawn up the plate via capillary action.
7. High-performance liquid chromatography (HPLC)
•used to separate and to purify proteins/peptides based on size,
charge or overall hydrophobicity.
•High-performance liquid chromatography (HPLC; formerly
referred to as high-pressure liquid chromatography), is a
technique in analytical chemistry used to separate, identify, and
quantify each component in a mixture. It relies on pumps to pass
a pressurized liquid solvent containing the sample mixture through
a column filled with a solid adsorbent material.
•Each component in the sample interacts slightly differently with
the adsorbent material, causing different flow rates for the different
components and leading to the separation of the components as
they flow out the column.
Two-dimensional (2-D) gel electrophoresis
A powerful gel-based method commonly used for
‘global’ analysis of complex samples
In this technique the protein is run first in a narrow (often tube-
shaped) isoelectric focussing gel, which as already noted
separates proteins on the basis of their isoelectric points (acid vs.
•The first step in separating large numbers of proteins in two dimensions
is to separate them according to their inherent charge
•The mixture of proteins is loaded onto a gel that has a gradient of
•An electric field is applied and the proteins move along the pH gradient
until they reach the point at which their charges are neutralized
•At this point, each band in the gel contains several different proteins with the same
(or very similar) isoelectric point. The tube gel is removed from its tube and exposed
to SDS to denature the proteins
•It is then placed on a slab of polyacrylamide gel and traditional SDS-PAGE is run in
the second dimension to separate the proteins by size. After staining, the result of 2D-
P AGE is a square with small scattered dots representing individual proteins
11. Father of Electrophoresis -Arne W.K. Tiselius
(Swedish physical biochemist , 1902-1971)
The Nobel Prize in Chemistry 1948 for the discovery of proteins in
blood serum and for the development of electrophoresis as a technique
for studying proteins.
Gel electrophoresis is one of the most basic important tools used by the
molecular biologists in the study of DNA or its fragments.
Gel electrophoresis technique is used-
To study the purity and intactness of DNA
To separate and identify the fragments
To analyze and characterize recombinant DNA molecules.
12. Gel electrophoresis is a method that separates macro molecules- either nucleic
acids or proteins-on the basis of size, electric charge, molecular mass and other
Gels are used for 2 reasons:
Gels suppress convection current produced by small temperature gradients, a
requirement for effective separation and
Gels resolve as molecular sieves that enhance the separation.
Migration of a molecule is inversely proportional to its frictional coefficient
while frictional coefficient is dependent on size and shape of the molecule. But
DNA molecule can assume different conformations (shapes) and this fact
complicates electrophoretic analysis of DNA.
13. Many important biological molecules such as amino acids, peptides,
proteins, nucleotides, and nucleic acids, posses ionisable groups and,
therefore, at any given pH, exist in solution as electrically charged
species either as cations (+)or anions(-).
Depending on the nature of the net charge, the charged particles will
migrate either to the cathode or to the anode.
The rate of this electrophoretic migration or mobility depends on the
pH of the medium, strength of the electric field, magnitude of the net
charge on the molecule and the size of the molecule
14. AGAROSE GELS
•For the separation of :
(1) large protein or protein complex
(2) polynucleotide 50-30,000 base-pairs
The pore size is determined by adjusting the concentration of agarose
in a gel (normally in the rank of 0.4-4%)
16. ELECTROPHORETIC APPARATUS
A power pack -to supply uninterrupted constant current.
Electrophoresis unit: Gel casting tray -to cast theAgarose gel.
Plastic combs -to form loading wells for sample in the gel.
Electrophoresis tank -to pour buffer, (usually TAE/TBE) provide ions to support conductivity.
DETECTION OF BANDS
Detect bands by staining during or after electrophoresis
Ethidium bromide: for double-stranded DNA
SyBr green or SyBr gold: for single- or double-stranded DNA or for RNA.
Silver stain: more sensitive for single- or double-stranded DNA or for RNA and
•Determination of DNA sequences.
•Southern and Northern blotting.
•Restriction mapping of DNA.
•Determination of subunit of protein.
•Determination of molecular weight of protein
•Poly acrylamide gels are the supporting media of choice for electrophoresis because they are
•Formed from the polymerization of acrylamide monomer in the presence of N, N’- methylene bis
•Polymerization is initiated by the addition of ammonium per sulphate (APS) &base tetra methylene
diamine (TEMED). TEMED catalyses the decomposition of persulphate ion to give a free radical.
•O2 removes free radicals therefore the solutions are placed under vacuum to remove loosely
dissolved O2 before use.
•Pore size-varied by changing the concentration of acrylamide & bis-acrylamide.
19. Polymerization should take place under an inert atmosphere since oxygen can act as a
free radical trap
The polymerization is temperature dependent : to prevent incomplete polymerization
the temperature should be maintained above 20⁰c.
To minimize oxygen absorption gels are usually polymerized in vertical casting
The gel is divided into two areas: resolving and stacking gel.
The resolving gel with small pores of pH 8.8 , the stacking gel with large pores of pH
6.8. (to concentrate the protein sample before it enters resolving gel).
•In this method of electrophoresis, the polyacrylamide gel contains 0.1% SDS, and the
proteins are treated with an excess of SDS ( sodium dodecyl Sulphate), which is anionic
(negatively charged) detergent.
20. SDS has the following effect on protein molecules:
1.all hydrogen bonds are broken,
2.all hydrophobic interactions are cancelled,
3.aggregation of the proteins is prevented,
4.individual charge differences of the proteins are masked, and
5.the polypeptides become unfolded (removal of tertiary structure)
***However, SDS does not affect disulphide bonds and hence β-
mercaptoethanol is used.(samples are boiled for 5min in buffer sample
containing both SDS and βmercaptoethanol )
Estimation of the size of DNA molecules following
restriction enzyme digestion, e.g. in restriction
mapping of cloned DNA.
Analysis of PCR products, e.g. in molecular genetic
diagnosis or genetic fingerprinting.
Separation of restricted genomic DNA prior to
Southern transfer, or of RNA prior to Northern
25. why do we do protein analysis?
Isolated proteins are often used in foods as
ingredients because of their unique functional
Quantitative proteomics includes powerful global
discovery or targeted methods to analyze and
understand protein dynamic changes in cells or
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.
26. By conducting a study on Gel electrophoresis and the types of gels
(agarose and polyacrylamide gels) used for this technique, we
understand the various principles and theory behind performing this
We also learnt its importance in the study of the genetic materials
(especially DNA and its fragments), which are the basic elements of all
RNA and other important proteins are also studied with the help of gel