Sodium dodecyl sulphate - Polyacrylamide Gel Electrophoresis (SDS - PAGE) is a technique used for the separation of deoxyribonucleic acid (DNA) ,Ribonucleic acid (RNA) And protein molecules according to their size and electrical charge.

1. Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis
TOPIC-
SDS-PAGE Electrophoresis
Presented By – Dimple Gupta

2. GEL ELECTROPHORESIS
It is a technique used for the separation of Deoxyribonucleic
acid, Ribonucleic acid or protein molecules according to their
size and electrical charge using an electric current applied to
a gel matrix.
What is a gel?
Gel is a cross linked polymer whose composition and porosity
is chosen based on the specific weight and porosity of the
target molecule.
Types of gels used: Polyacrylamide gel, Agarose gel.

3. • Polyacrylamide gels which were first used for electrophoresis by
Raymond &Weintraub (1959) are chemically inert and particularly stable.
• SDS-PAGE is most widely used method for qualitatively analyzing protein
which is based on the separation of protein according to the size.
• SDS-PAGE is the most commonly used gel electrophoretic system for
analyzing proteins.
• Method is based on the separation of proteins according to size and can
also be used to determine the relative molecular mass of proteins.

4. Why SDS?
SDS is an anionic detergent which binds strongly to and
denatures proteins to produce linear polypeptide chains. The
presence of β-mercaptoethanol assists in protein denaturation
by reducing all disulfide bonds. The detergent binds to the
hydrophobic region of the denatured protein in a constant ratio.
The protein-SDS complex carries net negative charges, hence
move towards the anode and the separation is based on the size
of the proteins. The protein-SDS complex carries net negative
charges, hence move towards the anode and the separation is
based on the size of the proteins.
SDS solubilize cell membrane of the
biological sample.
SDS (sodium dodecyl sulfate)

5. PAGE (polyacrylamide gel electrophoresis )
PAGE describes a
technique widely used in
biochemistry ,forensics,
genetics,molecular
biology, biotechnology,
to seprate biological
macromolecules ,usually
proteins or nucleic acids
Acc. to their
electrophoretic mobility.

6. Principle
 SDS-PAGE (Polyacrylamide Gel Electrophoresis), is an analytical method used to
separate components of a protein mixture based on their size.
 The technique is based upon the principle that a charged molecule will migrate in an
electric field towards an electrode with opposite sign. The general electrophoresis
techniques cannot be used to determine the molecular weight of biological molecules
because the mobility of a substance in the gel depends on both charge and size.
 To overcome this, the biological samples needs to be treated so that they acquire uniform
charge, then the electrophoretic mobility depends primarily on size. For this different
protein molecules with different shapes and sizes, needs to be denatured (done with the
aid of SDS) so that the proteins lose their secondary, tertiary or quaternary structure .The
proteins being covered by SDS are negatively charged and when loaded onto a gel and
placed in an electric field, it will migrate towards the anode (positively charged electrode)
are separated by a molecular sieving effect based on size. After the visualization by a
staining (protein-specific) technique, the size of a protein can be calculated by comparing
its migration distance with that of a known molecular weight ladder (marker).

7. INSTRUMENTATION
• Equipment required for electrophoresis basically consists of two items
• An electrophoresis unit
• A power pack
• Electrode chamber -upper chamber and a lower chamber. Both chamber are fitted
with the platinum electrodes
• Power supply- supplies a direct current between the electrodes in the
electrophoresis unit.

8. Contd.
• Glass plates - rectangular and notched
• The gel is formed between two glass plates, clamped together but held apart by
plastic spacers.
• Gel dimensions are typically 12cmX 14cm, with a thickness of 0.5 to 1mm.
• A plastic comb is placed in the gel solution and is removed after polymerization to
provide loading wells for samples.
• Casting frame
• Casting stand
• Running Module
• Platform shaker

9. Buffer and Reagent of SDS-PAGE
 Chemical buffer: It Stabilizes the pH value to the desired value within the gel itself and in
the electrophoresis buffer. Common buffers in PAGE include Tris, Bis-Tris, or imidazole.
 Tris-HCl- It is the component of running and gel casting buffer
 Acrylamide (C3H5NO) : It is monomeric unit used to prepare the gel.
 Bisacrylamide (N,N′-Methylenebisacrylamide) (C7H10N2O2 ): It is used for cross
linking agent for polyacrylamide gels. Bisacrylamide can crosslink two polyacrylamide
chains to one another, thereby resulting in a gel.
 Sodium Dodecyl Sulfate (SDS) (C12H25NaO4S): SDS is a strong detergent agent used
to denature native proteins to unfolded, individual polypeptides. It gives a near uniform
negative charge along the length of the polypeptide.
 Ammonium persulfate (APS) (N2H8S2O8 ): APS is a source of free radicals and is used
as an initiator for gel formation.
 TEMED (N, N, N′, N′-tetramethylethylenediamine) (C6H16N2 ): TEMED stabilizes free
radicals and improves polymerization. The rate of polymerisation and the properties of the
resulting gel depend on the concentrations of free radicals.

11. Chemicals used for Processing & Visualization
 Tracking dye As proteins and nucleic acids are mostly colorless. A very common
tracking dye is Bromophenol blue
(BPB, 3',3",5',5" tetrabromophenolsulfonphthalein).
 As it reaches the anodic end of the electrophoresis medium electrophoresis is
stopped. It can weakly bind to some proteins and impart a blue colour.
 Other common tracking dyes are xylene cyanol, which has lower mobility, and
Orange G, which has a higher mobility.
 Coomassie Brilliant Blue R-250 (CBB)(C45H44N3NaO7S2): CBB is the most
popular protein stain. It is an anionic dye, which non-specifically binds to proteins.
 Destaining Solution: 15% (v/v) Methanol and Acetic Acid (10% v/v) in Triple Distilled
water. The excess dye incorporated into the gel can be removed by destaining with the
same solution without the dye. The proteins are detected as blue bands on a clear
background.

12. Sample Preparation
A protein sample
• Samples may be any material containing proteins. These may be
biologically derived, from prokaryotic or eukaryotic cells, tissues, viruses,
environmental samples, or purified proteins.
• Heat at 95 C
Loading buffer
• The sample is prepared in the loading dye containing
• Tris HCl pH-6.8
• SDS (sodium dodecyl sulfate)
• Β-mercaptoethanol
• glycerol to denature the sample and presence of glycerol facilitates the loading of
sample in the well.

14. Casting of the gel
Resolving Gel Stacking Gel

15. Resolving gel
 Resolving gel is also called as
separating Gel
 It creates Smaller pores,higher ionic
strength
 Polyacrylamide gel is made using a pH
8.8 Tris/HCl buffer.
 In the resolving gel, macromolecules
separate according to their size.
 Resolving gels have an optimal range of
separation that is based on the percent
of monomer present in the
polymerization reaction. After adding TEMED and APS to the SDS-
PAGE separation gel solution, the gel will
polymerize quickly so add these two
reagents when ready to pour.

16. Stacking gel
 Stacking gel is creates Larger pores, &
lower ionic strength
 This gel is prepared with Tris/HCl buffer
pH 6.8 of about 2.0 pH units lower than
that of electrophoresis buffer
(Tris/Glycine).
 Pour stacking gel on top of the separation
gel and comb is fitted into the gel for
construction of different lanes for the
samples
1M Tris pH6.8
The polymerized gel is known as the “gel cassette”.

17. Stacking Gel Vs Resolving Gel

18. Running Buffer
 Dissolve 30.0 g of Tris base,
 144.0 g of glycine
 10.0 g of SDS in 1000 ml of H2O.
 The pH of the buffer should be 8.3
 Glycine is an amino acid whose
charge plays an important role in
the stacking gel.

19. PROCEDURE

20. PROCEDURE
• Clean the glass plates and
spacers of the gel canting
unit with deionized water
and ethanol
• Assemble the plates with
the spacers on a stable,
even surface.

21. Casting Of Gel
• Take the Resolving Gel in Micropipette.
• Pour the Resolving gel solution in between the glass plate fitted into
the gel caster, Until the resolving gel Reaches just above the green
line or approx. 1cm from the top of the Plate.
• A thin layer of organic solvent (such as butanol or isoproponal) is
layered to stop the entry of oxygen and make the top layer smooth.
• Allow the gel to set for about 20-30 min at room temperature for
polymerization.
• After the polymerization of Gel, Drain the Organic Solvent.

22. After polymerization of the resolving gel, a stacking
gel is poured on the Top of the resolving Gel in
between the plates.
Fill to the Top, Then Insert the comb on the top of
stacking gel to enables the formation of wells in the
gel.
Wait for the gel is completely polymerize.
After the gel is formed, Remove the glass plates
from casting frame.

24. Running of gel
• Placed the Gel glass Plate onto the Running Module.
• Insert the Running Module Into the Electrophoresis Chamber.
• Pour the 10X Dilutyed Running buffer Solution into the Running module
Between the Two plates.
• It is essential to maintain a constant state of ionization of the molecules
being separated.
• Remove The Comb to generate the wells in the gel.

25. Running of gel
• Load 10 microliters of Sample Solution into Each
Well & equal amount of concentred protein to be
loaded in each well.
• For Molecular weight marker, A Standard solution is
also laoded into the one of the Lane.
• When All the samples were loaded.
• Close the lid on the Tank.
• Insert the Electrodes Properly Into the Power supply
& Set the Power for Run the Gel.

26. • Initially Observe The sample or Standard Solution to
be Condensed into a thin band Through The stacking
gel, Then the band will Gradually Migrate Down the
resolving Gel.
• When bromophenol blue Dyes have reached close to
the bottom of the Gel, Stop the Run.
• Unplug the Power supply, Remove the runing module
from the tank & Drain The Running Buffer.
• Remove the Glass Plate from the Running Module,
then use the Gel releaser to Remove the Gel from the
glass plates.

27. Protein Staining
• Transfer the Gel into a Square PetriDish.
• Pour the Coomassie Brilliant Blue R250 staining
solution into the petri dish, submerging the gel
fully.
• Agitate slowly on a platform shaker for 30-60
mins.
• CBB is a non-specifically stains
• It Stains almost all proteins in the Gel.

28. • Discard staining solution and wash
the gel with triple distilled water.
• Add 5 to 10 gel volume
of destaining solution.
• Agitate slowly on a platform shaker
for 30-60 mins.
• If the color of the destaining
solution is intense blue, replace it
with the new destaining solution.
• After Destaining, Distinct bands
appears within the gel.

29. Results
• In the beginning, whole gel will appear blue/black but the dye will be removed from
the background and give discrete appearance of protein bands.
• Different Proteins were observed in the gel.
• When we stain the gel with Bromophenol blue & allow it to move proteins
from cathode to Anode When power is supplied.

31. Image Acquisition
• The image acquisition in typical gel
documentation has multiple steps as
• A suitable staining of the protein bands within
the gel is analysed by 3 factors are-
 Position of Band
 Distance from the origin
 Intensity of band
 Intensity of stained band is directly proportional
to the amount of protein in a band.
Preview the gel bands and optimize parameters such as camera focus, brightness
and capture time, magnification, spatial resolution etc.
Once all parameters are optimized.
Its shows good quality image & image can be captured.
Save the image in a suitable digital image format.

32. APPLICATIONS
Measuring molecular weight
Used in peptide mapping
Estimation of protein purity
Estimation of protein size
Used in western blotting and protein ubiquitination
Used in HIV protein separation
Indivisual protein can be determined from a mixture
Widely used technique in forensics, biochemistry, molecular biology for
separation of macromolecules

33. Applications
SDS-PAGE is a reliable method for determining
the molecular weight (MW) of an unknown protein.
For that we need to first know the estimation of
molecular weight
 Calculate distance travelled for each band
 For each band in the standards, calculate the
Rf value using the following equation:
 Rf = migration distance of the
protein/migrationdistance of the dye front
 Repeat this step for the unknown bands in the
samples.
 Use a graphing program, plot the log (MW) as a
function of Rf.
 Generate the equation y = mx + b
DETERMINATION OF MOLECULAR
WEIGHT

34. After separation, determine the
relative migration distance (Rf) of the
protein standards and of the unknown
protein
The plot should be linear for most
proteins, provided they are fully
denatured and that the gel percentage
is appropriate for the MW range of the
sample.
The standard curve is sigmoid at
extreme MW values because at high
MW, the sieving effect of the matrix is
so large that molecules are unable to
penetrate

35. PEPTIDE MASS
FINGERPRINTING
Peptide mass fingerprinting or peptide
mapping is a technique to cleave
unknown target protein into smaller
peptides and getting absolute mass of
the peptide
Helps in identification of protein by
mass by charge ratio
Enzymatic treatment of protein leads to
smaller fragments

36. Western blotting and protein separation
• Western blot is a technique used
to detect protein in a sample
• Eg- HIV protein separation
• HIV makes DNA using reverse
transcription
• HIV proteins separated using
SDS PAGE and membrane
incubated with patient serum.
Antibodies bind to viral
proteins(capsid)
• Western blots provide early
detection of proteins

37. Advantages
• Stable chemically cross-linked gel
• Greater resolving power (Sharp bands)
• Can accommodate larger quantities of DNA
without significant loss in resolution
• The DNA recovered from polyacrylamide gels is
extremely pure
• The pore size of the polyacrylamide gels can be
altered in an easy and controllable fashion by
changing the concentrations of the two
monomers.
• Good for separation of low molecular weight
fragments

38. Disadvantages
• Generally more difficult to prepare and handle, involving a
longer time for preparation than agarose gels.
• Toxic monomers
• Gels are tedious to prepare and often leak
• Need new gel for each experiment Stable chemically cross-
linked gel

39. Advancements in sds PAGE
• 2-D gel sds PAGE
• 2-DE consists mainly of two steps of separation; first
dimension and second dimension. In the first
dimension, protein molecules are resolved depending
on their isoelectric point
• 2-DE is a powerful and widely used method for
analysis of complex protein mixtures with exceptional
ability to separate thousands of proteins at once.
• Upto 5000 proteins can be observed.
• Resolves the problem of optimal resolution using extra
filter
• Proteins form zwitter ion at isolectric point(net charge
0)
• Different protein at different pH settle at their isoelectric
point

40. 2 DIGE ELECTROPHORESIS
• differential imaging gel electrophoresis. This method was designed in an attempt
to increase sensitivity and reproducibility of 2-DE using multiplexed fluorescent
dyes- labeled protein samples
• Shows gel to gel variation
• different fluorescent cyanine (Cy) dyes are used for labeling proteins from
different samples

41. Reference
• www.ncbi.nlm.nih.gov
• Sds page biology with animation
• https://www.ijsr.net
• https://capricorn.bc.edu
• https://experiments.springernature.com
• https://nptel.ac.in/content