Here I have Explained About Polyacrylamide Gel Electrophoresis . What is The Principle of Electrophoresis? What is Gel Electrophoresis? Types of Gel Electrophoresis. What is PAGE? How PAGE Run? Why SDS PAGE?

1. PAGE
(Polyacrylamide gel
electrophoresis)
Submitted By – Omedul Mondal
3rd Semester M.Sc. Applied Genetics.
Subject - MGE- 301: Genetic Engineering
Reg. No.-20IAMOS007
Submitted to – Professor Subha Rajiv
Department of Genetics
Indian Academy Degree College
Department of Genetics

2. CONTENTS
• What is Electrophoresis and Its Principle?
• What is Gel Electrophoresis? Types of Gel Electrophoresis
• What is PAGE?
• How PAGE runs?
• Why SDS PAGE?
• Difference between PAGE and Agarose Gel?
• What is the

3. INTRODUCTION OF ELECTROPHORESIS
• Migration of a charged particle under the influence of an electric
field.
• Word Electrophoresis means “to carry with electricity”
• Arne Tiselius(father of electrophoresis), with support from the
Rockefeller Foundation, developed the "Tiselius apparatus" for
moving boundary electrophoresis, which was described in 1937 in the
well-known paper "A New Apparatus for Electrophoretic Analysis of
Colloidal Mixtures“.

4. FATHER OF ELECTROPHORESIS

5. PRINCIPLE

6. According to law of electrostatics, an ion with charge ‘Q’ in an electric field of
strength ‘E’ will experience an electric force
Resulting migration of the charged molecule through the solution is opposed by
a frictional force
Frictional coefficient depends on size and shape of the migrating molecule and
viscosity of the medium.
Felectrical=Q.E
Ffrictional=V.f [V =rate of migration of charged molecule;
f=frictional coefficient]
f=

7. In the constant electric field the force on charged molecule balances each
other
The migration of the charged molecule in the electric field is termed as
electrophoretic mobility( ) which is the ratio of the migration rate of a charged
molecule to the applied electric field
Electrophoretic mobility is directly proportional to the charge and inversely
proportional to the viscosity of the medium, size and shape of the molecule.
QE=Vf
=Qf=VE

8. Gel Electrophoresis
• Gel electrophoresis is a method for separation and analysis of
macromolecules (DNA, RNA and proteins) and their fragments, based
on their size and charge.
• Used in clinical chemistry to separate proteins by charge or size and
in biochemistry and molecular biology to separate a mixed population
of DNA and RNA fragments by length, to estimate the size of DNA and
RNA fragments or to separate proteins by charge.

9. Gel
Electrophoresis
Agarose gel
Electrophoresis
Starch Gel
Electrophoresis
Poly Acrylamide
Gel
Electrophoresis

10. Polyacrylamide gel
• Consist of chains of acrylamide monomers crosslinked with
Bisacrylamide ( Two units of acrylamide connected by
methylene bridge).

11. • Polymerization of acrylamide is initiated by the quaternary amine TEMED(
N,N,N,N-tetramethylethylenediamine ) which induces free radical
formation from ammonium persulphate(APS).
• Acrylamide and Bisacrylamide are a neurotoxin in their monomer form.
• Resolving power and pore size of a gel depends on the concentration of
acrylamide and bisacrylamide.
• Lower percentage gel are better for resolving very high molecular weight
molecule, higher percentage resolve small molecule.
• Protein Mol.Wt range <5k to >200k and polynucleotide from <5 to 3k bp in
size.
• Gels that have single acrylamide percentage(concentration) throughout the
gel :- Uniform Acrylamide concentration gel.
• Gels that have different acrylamide percentage through the gel ,a low % of
acrylamide at top and high % of acrylamide at bottom:- Gradient
Acrylamide Gels.

12. Buffer System
1. Gel casting buffer – To cast the gel.
2. Sample Buffer- To prepare sample.
3. Running Buffer – To fill the electrode reserviour.

13. PAGE

14. SDS PAGE
• Relative Movement of protein through PAGE depends on Charge
Density(charge per unit mass), mass(or size) and the shape of the
molecule.
• In SDS PAGE proteins are treated with negatively charged anionic
detergent sodium dodecyl sulfate before and during gel
electrophoresis.
• SDS imparts large net negative charge on proteins.
• In SDS-PAGE migration of proteins are not determined by intrinsic
electric charge of polypeptide but by molecular weight because SDS
treatment eliminates the effect of difference in charge density and
shape.

15. • Therefore in when current is on all SDS bound proteins in a sample
will migrate towards positively charged electrode; proteins of less
mass travel more quickly than those with greater mass.
• In reducing SDS PAGE, along with SDS protein is treated with
reducing Agents such as β-mercaptoethanol, dithiothreitol to break
intrachain or interchain disulfide bonds between cysteine residues.
• Nonreducing SDS PAGE reducing agents are not used.

16. Native PAGE
• In native PAGE, proteins are separated according to the net charge,
size and shape of their native structure.
• Most protein molecules carry a net charge at any pH other than their
isoelectric point and hence electrophoretic migration occurs because
most proteins carry a net negative charge in alkaline running buffers.
• The higher the negative charge density the faster a protein will
migrate. At the same time the frictional force of the gel matrix creates
a sieving effect, regulating the movement of proteins according to
their size and three dimensional shape.
• Small proteins face only a small frictional force, while larger proteins
face a larger frictional force.

17. • Thus, in native PAGE , proteins are prepared in nonreducing,
nondenaturing sample buffer, and electrophoresis is perfomed in the
absence of denaturing and reducing agents.
• Native charge is preserved , separates proteins based upon their charge,
mass and shape in their native state and can resolve proteins of the
same molecular weight.

18. Sample Visualization in the Gel
• After electrophoresis run is complete ,proteins are visualized by gel
staining.
• Coomassie brilliant blue(the lower detectablelimit about 0.1-0.5microgram
protein) is the most widely used dye which binds to proteins but not to gel
itself. In acidic buffer condition Comassie dye binds to basic and
hydrophobic residues of proteins, changing in colour from dull reddish-
brown to intense blue.
• Silver staining(lower detection limit about 0.5ng of proteins) is the most
sensitive method for permanent visible staining of proteins in
polyacrylamide gels. Here, silver ions is deposited to give a brown black
color. Silver ions interacts and binds with carboxylic acid groups(Asp & Glu),
imidazole(His), Sulfhydryls(Cys) and amines(Lys).

19. Estimating the molecular weight of a protein

20. Immunoblotting

21. PROCEDURE

22. SET UP GEL
Get The purified
Sample
Load Sample
Run Gel
Stain and look at UV Light

23. Assembling the glass plates:
• Assemble the glass plate on clean surface. Lay the longer glass plate
(the one with spacer) down first,then place the shorter glass plate on
top of it.
• Embed them into the casting frame and clamp them properly make
sure that the bottom ends of the glass plates are properly aligned
• Then place it on the Casting Stand.

25. Casting the Gels
• Prepare 10% of resolving gel .
• Prepare the separating gel solution by combining all reagent.
• Add APS and TEMED to the monomer solution(just before pouring) and mix
well by swirling gently. Pour the solution till the mark.
• Allow the gel to polymerize for 20-30minutes.
• Prepare 4.5% stacking gel. Drain the isopropanol with strips of filter paper.
• Add APS and TEMED to the monomer solution and mis well by swirling
gently.
• Place a comb in the stacking gel sandwich. Allow it to polymerize for 10
minutes.

26. Preparation of Samples
• Mix your protein in the ratio 4:1 with the sample buffer. Heat your
sample by either: (a) Boiling for 5-10minutes. (b) 65 C for 10 minutes.
(c) 37 C for 30 minutes

31. References
• Kumar P; Biophysics and Molecular Biology Fundamentals and
Techniques; Pathfinder Publication; Third Edition; New Delhi
• Wilson.K and Walker.J ;Principles and Techniques of Biochemistry and
Molecular Biology; Cambridge University Press; Seventh Edition; UK
•