Electrophoresis of Proteins

1. Sodeum dodecyl sulphate-Sodeum dodecyl sulphate-
Polyacrylamide gel electrophoresisPolyacrylamide gel electrophoresis
(SDS-PAGE)
Dr Ravi Kant Agrawal, MVSc, PhD
Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India

2. IntroductionElectrophoresis: Migration of a charged particle under the influence of electric
current. A method of separating large molecules (such as DNA fragments or
Proteins ) from a mixture of similar molecules.
 An electric current is passed through a medium containing the mixture, and
each kind of molecule travels through the medium at a different rate,
depending on its electrical charge and size.
 Separation is based on these differences.
 Agarose and acrylamide gels are the media commonly used for
electrophoresis of proteins and nucleic acids.
 Agarose is used for nucleic acids while polyacrylamide is used to separate
proteins/ small oligonucleotides
 Proteins are versatile molecules with diverse functions may be structural or
non- structural, like enzymes.
 To study protein function, it should be in pure form.
 Proteins are amphoteric compounds and their net charge is determined by
the pH of the medium.
 Many supporting media are used like cellulose acetate paper, silica gel,
agarose or polyacrylamide.
 Pores of agarose gels are larger as compared to polyacrylamide.

3. Gel Electrophoresis
• The pH and other buffer conditions are arranged so that the
molecules being separated carry a net (negative) charge so that
they will be moved by the electric field toward the positive
pole.
• As they move through the gel, the larger molecules will be held
up as they try to pass through the pores of the gel, while the
smaller molecules will be impeded less and move faster.
• This results in a separation by size, with the larger molecules
nearer the well and the smaller molecules farther away.

5. Strength of electric field
Ionic strength (pH variation of buffer), viscosity and
temperature of medium in which
protein is moving.
Net charge, size, shape of protein molecules.
Support medium’s pore size.
Factors affecting the rate of migration

6. Powerful electrophoretic techniques developed to separate
macromolecules on the basis of molecular weight.
 The mobility of a molecule in an electric field is inversely
proportional to molecular friction which is result of its
molecular size and shape, and directly proportional to voltage
and charge of molecule.
In PAGE, proteins charged negatively by the binding of the
anionic detergent SDS (sodium dodecyl sulfate) separate within
a matrix of polyacrylamide gel in an electric field according to
their molecular weights.
Principles of Polyacrylamide Gel
Electrophoresis

7. Contd..
• Polyacrylamide gels are made from polymerization of
Acrylamide and Bisacrylamide where Acrylamide forms linear
strands and cross linking of the strands is brought by
bisacrylamide.
 Acrylamide + N N’ methylene bis
acrylamide
Polyacrylamide
Ammonium persulfate
(catalyst) + TEMED
Chemical
Polymerizatio
n

9. Polyacrylamide Gel Electrophoresis
• Monomeric acrylamide (which is neurotoxic) is polymerized in the
presence of free radicals to form polyacrylamide.
• The free radicals are provided by ammonium persulphate and
stabilized by TEMED (N'N'N'N'-tetramethylethylene-diamine).
• The chains of polyacrylamide are cross-linked by the addition of
methylenebisacrylamide (bis) to form a gel whose porosity is
determined by the length of chains and the degree of crosslinking.
• Polyacrylamide gels are poured between two glass plates held apart
by spacers of 0.4 - 1.0 mm and sealed with tape.
• Most of the acrylamide solution is shielded from oxygen so that
inhibition of polymerization is confined to the very top portion of the
gel.
• The length of the gel can vary between 10 cm and 1m depending on
the separation required.
• They are always run vertically with 0.5x or 1x TBE as a buffer.

10. contd.
Polymerization initiated Ammonium Persulphate (1.5%,W/v)
provides free radicals and TEMED (N,N,N,N-tetra-methylene –
ethylene-diamine) acts as activator of reaction.
Concentration of acrylamide and Bisacrylamide solution is
increased, the pore size of the gel is decreased.
Advantage of acrylamide gel systems is that initial
concentrations of acrylamide and BIS control the hardness and
degree of crosslinking of gel.
Generally resolving gels varying between 10-15% and stacking
gels of 5% are used.

11. Principle of SDS-PAGE
• SDS (also called lauryl sulfate) is an anionic detergent, meaning that when
dissolved its molecules have a net negative charge within a wide pH range.
• A polypeptide chain binds amounts of SDS in proportion to its relative
molecuar mass.
• The negative charges on SDS destroy most of the complex structure of
proteins, and are strongly attracted toward an anode (positively-charged
electrode) in an electric field.
• Polyacrylamide gels restrain larger molecules from migrating as fast as
smaller molecules.
• Because the charge-to-mass ratio is nearly the same among SDS-denatured
polypeptides, the final separation of proteins is dependent almost entirely
on the differences in relative molecular mass of polypeptides.
• In a gel of uniform density the relative migration distance of a protein (Rf,
the f as a subscript) is negatively proportional to the log of its mass.
• If proteins of known mass are run simultaneously with the unknowns, the
relationship between Rf and mass can be plotted, and the masses of
unknown proteins estimated.

13. Wash glass plates and spacers in warm detergent solution and
rinse with tap water and deionized H2O.
 Rinse plates with ethanol and dry it. The glass plates must be
free of grease spots to prevent air bubbles in gel.
Assemble the glass plates with spacers.
 Lay the larger (or unnotched) plate flat on the bench and
arrange the spacers at each side parallel to the two edges.
 Apply petroleum jelly to keep the spacer bars in position during
the next steps.
 Lay the inner (notched) plate in position, resting on the spacer
bars.
 Clamp plates together with paper clips and bottom of plates
with tape to make a watertight seal.
Prepare gel solution with desired polyacrylamide percentage,
which gives amount of each component required to make 100
ml.
How to run SDS-PAGE

14. Volumes of Reagents to Cast Polyacrylamide Gels of
Indicated Concentrations in 1x TBE
Polyacryla
mide gel
(%)
29% Acrylamide
plus
1% N,N´-
Methylenebisacryla
mide
H2O 5x TBE
Ammoniu
m
Persulfate
3.5 11.6 62.7 20.0 0.7
5.0 16.6
62.7 20.0 0.7
8 26.6 52.7 20.0
0.7
12.0 40 39.3
20.0 0.7
20.0 0.7

15. Contd.
Place the required quantity of acrylamide:bis solution in flask
with a magnetic stir bar.
De-aerate solution by applying vacuum, it reduce chance that air
bubbles will form when thick gels (>1 mm) are poured and
reduce amount of time required for polymerization.
Add 35 μl of TEMED for each 100 ml of acrylamide:bis solution,
and mix the solution by gentle swirling.
Gels can be cast with 1 μl of TEMED per milliliter of gel solution
to increase rate of polymerization.
Immediately insert comb into the gel, being careful not to allow
air bubbles become trapped under teeth.
Allow the acrylamide to polymerize for 30-60 minutes at room
temperature.

16. Contd..
After polymerization is complete, surround comb and top of gel
with paper towels soaked in 1x TBE.
Seal the entire gel and store it at 4°C until needed.
When ready to proceed with electrophoresis, squirt 1x TBE
buffer, pull comb from polymerized gel.
Use a syringe to rinse out wells with 1x TBE. Remove tape.
Attach gel to electrophoresis tank, using clips on sides.
Notched plate should face inward toward buffer reservoir.
Fill reservoirs of electrophoresis tank with TBE buffer.
Use syringe to flush out wells once more with 1x TBE. Mix the
DNA samples with the appropriate amount of 6x gel-loading
buffer.
Load the mixture into wells using micropipette.

17. Contd..
 Connect electrodes to a power pack (positive electrode
connected to the bottom reservoir), turn on the power, and
begin the electrophoresis run.
 Run the gel until the marker dyes have migrated the desired
distance.
 Turn off electric power, disconnect the leads, and discard the
electrophoresis buffer from the reservoirs.
 Detect the positions of bands of DNA in the polyacrylamide gel.

19. Pros of Polyacrylamide Gel Electrophoresis
 PAGE has a high loading capacity, up to 10 micrograms of DNA
can be loaded into single well without significant loss of
resolution.
 Polyacrylamide contains few inhibitors of enzymatic reactions.
 PAGE is an ideal gel system from which isolate DNA fragments
for sub cloning and other molecular biological techniques.

20. Cons of Polyacrylamide Gel Electrophoresis
 Mobility of fragments affected by base composition making
accurate sizing of bands a problem.
 Polyacrylamide quenches fluorescence, making bands
containing less than 25 ng difficult to visualize with staining.

21. Different types of Polyacrylamide gel
electrophoresis
Native PAGE: no detergent like SDS is used. used for native 3-D
structure of proteins, also called non-dissociating PAGE.
Discontinuous PAGE: When a stacking gel of low concentration
is used over separating gel.
Continuous PAGE: When no stacking gel is used.
Reducing PAGE: When reducing substance like β-
mercaptoethanol is used to break disulfide bonds in protein
sample.
Non-reducing PAGE: When no reducing agent is used.
SDS-PAGE: When SDS an anionic detergent used in sample
buffer as well as stacking and separating gels to unfold protein,
also called dissociating PAGE.
Preparative PAGE: used to collect particular protein band in
large quantity from the protein mixture by making a single wide
well at the top.

22. Uses forPAGE
Separate from other proteins
Proteins separated by size
Isoelectric point
Determines
Molecular size of protein
Quantifies the amount present
 Displays Impurities
 Used in western blot assays

23. Migration of molecular weight of
standards are compared to unknown
samplewt

24. Movement of Proteins on an SDS Gel
Stacking of proteins at top
of gel at start
Protein Migration
Highest
Molecul
ar Wt.
protein
Distribution of
proteins in a
charged field
Low weight
molecular dye
-
+

25. Vertical Polyacrylamide Gel Electrophoresis

26. Thanks
Acknowledgement: All the material/presentations available online on the subject
are duly acknowledged.
Disclaimer: The author bear no responsibility with regard to the source and
authenticity of the content.
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