1. Biological techniques assignment
Group members name:
Areej Ahmad Roll no Z-19 24.
Marryam Faiz. Roll no Z-18 15.
CLASS: BS Zoology 7th Semester.
Topic name: SDS-PAGE Electrophoresis (Sodium Dodecyl Sulfate polyacrylamide
gel)
Submitted to: Dr Hafiz Abdullah Shakir.
(Areej Ahmad -24)
History:
Raymond and Weintraub in 1959 introduced a polyacrylamide gel as a useful
stable, flexible gel for zone electrophoresis.
Definition:
“SDS-PAGE is a technique used to separate proteins according to their
electrophoretic mobility.”
Principle:
• Electrophoretic mobility is a function of the length, conformation and charge
of the molecule. When proteins are separated by electrophoresis through a gel
matrix, smaller proteins migrate faster due to less resistance from the gel matrix.
• Proteins are not negatively charged. For the separation of proteins using gel
electrophoresis, first mix the proteins with a Sodium dodecyl Sulphate (Detergent
with a strong protein denaturing effect). The detergent makes the protein unfold
into a linear shape and coats them with a negative charge, which allows them to
migrate toward the positive end of the gel and be separated.
2. Instrument set-up and chemicals:
Following instruments and chemicals required in SDS-PAGE:
• Electrophoresis tank
• Dc power supply
• Glass plates
• Casting frame
• Casting stand
• Spacers
• Combs
• Gel solution
• SSB (A mixture of SDS, buffer, B-mercaptoethanol, glycerol & tracking dye
Bromophenol blue)
• Protein Sample
• Coomassie Brilliant blue (CBB)
• Staining Solution (mixture of Coomassie Brilliant blue: 50% methanol and
10% acetic acid)
• Destaining solution (50% methanol and 10% acetic acid)
SDS-PAGE Electrophoresis to overcome the limitations of native PAGE systems,
Laemmle (1970) incorporated the detergent Sodium Dodecyl Sulfate (SDS) into a
discontinuous denaturing buffer system, creating what has become the most
popular form of protein electrophoresis, SDS PAGE.
“SDS PAGE is an electrophoresis method to separate proteins in their
denatured state. “
This technique is generally used in my biochemistry forensics genetics and
molecular biology for the separation of proteins depending on their electrophoretic
mobility. Polyacrylamide is useful for high resolution electrophoresis of small
molecules like proteins.
3. Role of SDS:
Sodium Dodecyl Sulphate is a synthetic organic compound having formula
CH3 (CH2)11SO4Na. It is an anionic surfactant which is part of several cleaning
and hygiene products. This molecule is an organosulphate and a salt. SDS is used
for the identification and isolation of protein molecules. In most proteins binding
of SDS to polypeptide chain gives an even distribution of negative charge per unit
mass, thus causing separation during electrophoresis.
Principle:
SDS gel electrophoresis systems are used to determine the number and size of
protein chains or protein subunit chains in a protein preparation. Initially protein
preparation is treated with an excess of soluble thiol (usually 2 mercaptoethanol)
and SDS. Under these conditions, the thiol reduces all disulphide bonds present
within and between peptide unit, while the SDS (an ionic or denaturing detergent)
binds to all regions of the proteins and disrupts most non covalent intermolecular
and intramolecular protein interactions.
SDS is an anionic detergent that unfolds proteins and provides them with extra
negative charges. The amount of the associated SDS molecules and therefore the
number of charges is proportional to the length of the polypeptide chain. The SDS
gel separate individual polypeptide chains according to their size.
The velocity of proteins is an inverse linear function of the logarithm of their
molecular mass.
These two components result in total denaturation of the proteins in the sample,
yielding unfolded, highly anionic polypeptide chains.
The anionic polypeptide chains are then resolved electrophoratically with in a
polyacrylamide gel saturated with SDS and the appropriate current carrying buffer.
The excess SDS is included in the gel to maintain the denatured state of the
proteins during the electrophoretic separation. The SDS coated polypeptide
(carrying approximately one SDS molecule per two amino acids) create a situation
in which the charge to mass ratio of all of the proteins in the sample is
approximately the same.
At this point, the intrinsic charge on the individual polypeptide chains is
insignificant as compared with the negative charge imposed on them by the
presence of SDS. The friction experienced by the population of molecules as they
4. migrate through the polyacrylamide matrix is now the major factor influencing
differences in their mobility as SDS page employees’ two buffer polyacrylamide
gel compositions in a single slab.
5. Stacking gel
The stacking gel has large pores. This gel is prepared with Tris/ HCL
buffered pH 6.8 of about 2.0 pH his units lower than that of the
Electrophoresis buffer.
Running gel or resolving gel:
The resolving gel is a small pore polyacrylamide gel (3-30%
acrylamide monomer) typically made using a pH 8.8 Tris/HCl buffer.
Material required:
Power supplies: it is used to convert the AC current to DC current.
6. Protein Samples: The Protein is diluted using SDS-PAGE Sample
buffer and boiled for 10 minutes. A reducing agent such as
dithiothreitol or 2-mercaptoethanol is also added to reduce the
disulphide linkages to prevent any tertiary Protein folding.
7. Running buffer: The protein Samples loaded on the gel are run in
SDS-PAGE running buffer.
Staining and detaining buffer: The gel is stained with Coomassie
Stain solution. The gel is then detained with the Destaining solution.
Protein bands are then visible under naked eyes.
Protein ladder: Protein ladder is used to determine the location of
protein of interest, based on molecular size.
Marryam Faiz (Z18-15)
6-Procedure:
Acrylamide mixed with bisacrylamide (BIS) forms a cross linked polymer network when polymerizing
agent, ammonium persulfate(APS), is added. TEMED (N, N, N, N’-tetramethylenediamine) catalyses
polymerization reaction by increasing production of free radicals by APS.
Acrylamide +BIS = polyacrylamide.
Polymerization reaction causes a gel due to bisacrylamide, usually about 1 part in 35 relatives to
acrylamide, which results in cross links between two polyacrylamide molecules. The ratio of acrylamide
to bisacrylamide may be different in particular situations. There is also variation in acrylamide
concentration of gel that may be 5% to 25%.
Larger proteins are resolved better by lower percentage gels, while smaller proteins are resolved by
much higher percentage gels.
7-Molecular weight markers:
Protein markers of defined molecular weight are run together with samples in same gel and used as a
reference by which mass of sample proteins is found. These are called molecular weight markers.
8. a-Sample preparation:
In case of solid tissues first break down mechanically using a blender (large volumes) using a
homogenizer (smaller volumes) by sonicator or by using cycling of high pressure.
In case of tissues or cells, combination of biochemical and mechanical techniques including
various types of filtration and centrifugation are applied to isolate different cell sections and
organelles before electrophoresis.
Protein Samples are denatured by heating in presence of a sample buffer having 1% SDS with or
without a reducing agent such as 2mM DTT,2 mercaptoetanol (BME).
Protein sample is mixed with sample buffer and boiled for 3 to 5 minutes at 60 degrees.
Mixture is cooled to room temperature.
Tracking dye may be added to protein solution.
Pipette into sample well of a gel.
Loading buffers also have glycerol that is why they are heavier than water and settle down to
bottom of buffer submerged well when added to gel.
Homogenizer or blender:
It is a piece of laboratory or industrial equipment used for the homogenization of various types of
materials such as tissue, plant, food, and many others.
Sonicator:
An apparatus used to subject a biological sample to ultrasonic vibration (sound energy) so as to
fragment the cells, macromolecules and membranes.
b-Gel preparation:
Acrylamide, Bisacrylamide, and a buffer with an
adjusted pH are mixed.
Solution is degassed under vacuum to prevent
formation of air bubbles during polymerization
or butanol may be added to resolving gel after it
is poured, as butanol removes bubbles and
makes surface smooth.
Source of free radicals and a stabilizer such as
ammonium persulfate and TEMED are added to
initiate polymerization.
Gels are polymerized between two glass plates
in a gel caster.
Comb is inserted at the top to create the sample
wells.
After gel is polymerized comb is removed now
gel is ready for electrophoresis.
9. 8-Buffers:
Various buffer systems are used in SDS-PAGE depending on nature of sample and experimental
objective. The Buffers used at anode and cathode may be same or different.
9-Running SDS PAGE:
An electric field is applied across the gel, causing the negatively charge proteins to migrate
across the gel towards the positive electrode.
Depending on their size, each protein will move differently through the gel matrix: short
proteins will move easily through the pores in the gel, while larger ones will have move with
difficulty.
After a set amount of time (usually a few hours, though this depends on the voltage applied
across the gel; protein migration occurs more quickly at higher voltages, but these results are
typically less accurate than at those at lower voltages) the proteins will travel depending upon
their size; smaller proteins will travel farther down the gel, while larger one will stay closer to
the point of origin.
Thus proteins will be isolated nearly based on size.
10-Protein molecular Weight markers
Protein molecular weight markers are run in outer lanes of gel for comparison so that relative molecular
weight of a protein can be assessed on a gel. A standard curve can be made from distances travelled by
each marker protein. The distance travelled by unknown protein is then plotted and molecular weight is
estimated from standard curve. Several kinds of protein molecular weight markers are used that are
labelled or prestained for various modes of detection. These are prereduced.
10. 11-Staining of proteins (visualization):
Following electrophoresis the gel may be stained through Comassie brilliant blue or silver stain to
visualise the separated proteins. Choice between them depends on the sensitivity of detection that is
required for your application.
Through Coomassie brilliant blue:
The first method involves saturating the
gel with a solution of acetic acid,
methanol and water containing
Coomassie brilliant blue R-250 dye. As the
methanol and acetic acid in the solution
work to fix the proteins with in the gel
matrix, Coomassie brilliant blue binds to
the proteins in the gel. The interaction
between the Coomassie dye and proteins
has been shown to be primarily through
arginine residues, although weak
interactions with tryptophan, tryosine,
phenylalanine, histidine, and lysine are
also involved.
Silver staining:
Silver staining is sensitive to about 10 ng of protein
contained in a single band on the acrylamide gel. Silver
staining begins by saturating the gel with a solution of Silver
nitrate. Next, a reducing agent is added to cause the
reduction of Ag ions to metallic silver, which precipitate on
the proteins in the gel and cause the appearance of protein
bands that are black in colour. Silver staining is technically
more difficult and is used only when extreme sensitivity is
required.
12-Chemical ingredients and their roles:
Two different groups used PAGE in electrophoresis in 1959 polyacrylamide gel was considered a
potential embedding medium for sectioning tissues since 1964.It has many Electrophoretical
characteristics to make it a versatile medium it is a synthetic, thermostable, transparent strong,
chemically relative in inert gel. It can be made with a large number of average pore sizes. The pore sizes
of a gel depend on two factors
1. The total amount of acrylamide present (%T)( T is equal to total concentration of acrylamide and
bis acrylamide monomer.
2. The amount of c linker (%C)(C is equal to bisacrylamide concentration ).
11. 3. Pore size decreases with increasing %T; with cross linking. 5% C gives the smallest pore size. Gel
material can bear high voltage gradients. It is suitable to various staining and Destaining methods.
Gel material can be broken to get isolated components or dried for autodiography and permanent
recording.
14-Chemicals for processing and visualization:
Chemicals used for visualization and processing are as follows:
Tracking dye:
As proteins are mostly colourless so anionic dyes of a known electrophoretic mobility are generally used
as tracking dye.
Bromophenol blue:
It is coloured at alkali and neutral pH. This small negatively charge molecule that travels towards
annode. It weakly binds towards certain proteins. It gives blue colour.
Coomassie brilliant Blue R-250:
It is commonly used to stain proteins.
15-Applications of SDS-PAGE
1. It is used to measure the molecular weight of the molecules.
2. It is used to estimate the size of the protein.
3. Used in peptide mapping
4. It is used to compare the polypeptide composition of different structures.
5. It is used to estimate the purity of the proteins.
6. It is used in Western Blotting and protein ubiquitination.
7. It is used in HIV test to separate the HIV proteins.
8. Analyzing the size and number of polypeptide subunits.
9. To analyze post-translational modifications.
Advantages:
SDS-PAGE is usually the first choice as an assay of purity due to its reliability and ease.
Polyacrylamide gel electrophoresis of SDS-treated proteins allows researchers to separate proteins
based on their length in an easy, inexpensive, and relatively accurate manner.
Disadvantages:
The limitation of SDS-PAGE resides in its deliberate denaturation of proteins prior to
electrophoresis.
12. Enzymatic activity, protein binding interactions, detection of protein cofactors cannot be
determined on proteins isolated by SDS-PAGE.
Conclusion:
There are different types of electrophoresis is used to separate different molecules on the basis of absence
and presence of supporting matrix. On the base of the presence and absence of Supporting matrix,
electrophoresis mainly divided into:
Moving boundary electrophoresis
Zone electrophoresis
These two types of electrophoresis further divided into different types of electrophoresis based on the types,
size, mass/charge ratio, use of molecules in laboratory. Electrophoresis is an important laboratory
technique, used in forensics to compare DNA, in medical laboratories to do genetic testing, and in
microbiology labs to identify microorganisms. In addition to analyzing proteins or DNA, electrophoresis is
also used to create purified samples of proteins.
