Polyacrylamide gel electrophoresis (PAGE) is a technique used to separate proteins based on their size and charge. PAGE uses a polyacrylamide gel with a tight matrix and small pore sizes, allowing for the separation of smaller proteins. Sodium dodecyl sulfate-PAGE (SDS-PAGE) is a common type that uses the detergent SDS to denature proteins and impart a uniform negative charge, allowing separation based solely on molecular weight. The gel consists of a stacking gel that concentrates proteins, and a resolving gel where separation occurs. Proteins are visualized after electrophoresis by staining.

1. Page 1
Polyacrylamide Gel
Electrophoresis(PAGE)
Mr. R. K. Lodha

2. Why Use
Polyacrylamide
Gels to
Separate
Proteins?
• Polyacrylamide gel has a tight matrix
• Ideal for protein separation
• Smaller pore size than agarose
• Proteins much smaller than DNA
– Average amino acid = 110 daltons
– Average nucleotide pair = 649 daltons
– 1 kilobase of DNA = 650 kD
– 1 kilobase of DNA encodes 333 amino acids =
36 kD

3. Why polyacrylamide used for
a gel?
⚫Chemically inert
⚫Electrically neutral
⚫Hydrophillic
⚫Transparent for optical detection

4. Acrylamide
Page 4
•
•
Acrylamide CF- C3H5NO
White odourless crystalline solid, soluble in water,
ethanol,ether & chloroform
•
•
•
Prepared on industrial scale by the hydrolysis
of acrylonitrile by nitrile hydratase
carcinogenic as well as Neurotoxic compound.
used in the manufacture of dyes, Waste water
treatment and other monomers

5. Polyacrylamide
Page 5
• Also called Cross-linked Polyacrylamide
• Polyacrylamide is not toxic
• Polyacrylamide is a cross-linked polymer of Acrylamide
• It is recommended to handle it with caution
• It is highly water-absorbent, forming a soft gel when hydrated
• Used in-
- Flocculate or coagulate solids in a liquid
- A subdermal filler for aesthetic facial surgery
- Polyacrylamide gel electrophoresis
- In soft contact lenses etc.

6. Polyacrylamide gel
Page 6
• It is a white odorless gel, soluble in water
•
•
•
After polymerization of acrylamide it get cross-linked
structure
TEMED stabilizes free radicals and improves
polymerization
Here, the toxic affect of acrylamide get vanish (95%)
• Amount of polyacrylamide salt dissolved (conc.) is
directly proportion to cross –linked nature of gel

7. ▶Polyacrylamide gels are
characterized by two parameters:
1. total monomer concentration
(%T, in g/100 ml)
2. weight percentage of cross
linker (%C).
▶ Higher %T - smaller pores.
▶The practical ranges for
monomer concentration are
stock solutions of 30-40%

8. Gel Types
Page 8
•
•
•
•
•
•
•
•
•
•
•
Agarose
Polysaccharide extracted
from sea weed.
Gel casted horizontally
Non-toxic.
Separate large molecules
Commonly used for DNA
separations.
Staining can be done before
or pouring the gel.
Polyacrylamide Gel
• Cross-linked polymer of
acrylamide.
Gel casted vertically.
Potent neuro-toxic.
Separate small molecules.
Used for DNA or protein
separations.
Staining can be done after
pouring the gel.

9. A. On The Basis of Supporting Media
1.Slab Gel
A.Horizontal
B.Vertical
2. Tube Gel
B. On The Basis of Types of Separation
1. No-Denaturing/native (Separation by size and charge;
charge/mass and shape)
2. Denaturing/Non-Native (separation by size)
3. Other Types (IEF, 2-D-GEL)
TYPES OF GEL ELECTROPHORESIS

10. PAGE
Page 10

11. Poly Acrylamide Gel
Electrophoresis
Page 11
• It is a subtype of the gel electrophoresis
whereby the normal gel is replaced with
polyacrylamide gels used as support matrix.
• Gels are made by free radical-induced
polymerization of acrylamide and N,N’-
Methylenebisacrylamide.
• It is the most widely used technique of
electrophoresis.

12. Types Of PAGE
Page 12

13. •
•
•
No denaturing agents
Proteins separated based on size, charge and
shape.
Used when want to keep protein active to study
conformation, self-association or aggregation, and the
binding of other proteins
Native PAGE
Page 13

14. Principle
Electrophoretic migration occurs because most proteins
carry a net negative charge in alkaline running buffers.
The higher the negative charge density (more charges
per molecule mass), the faster a protein will migrate.
At the same time, the frictional force of the gel matrix
creates a sieving effect, retarding the movement of
proteins according to their size and three-dimensional
shape.
Small proteins face only a small frictional force while
large proteins face a larger frictional force.

15. Thus native PAGE separates proteins based upon both
their charge and mass.
Because no denaturants are used in native PAGE,
subunit interactions within a multimeric protein are
generally retained and information can be gained about
the quaternary structure.
In addition, some proteins retain their enzymatic
activity (function) following separation by native
PAGE. Thus, it may be used for preparation of purified,
active proteins.

16. Type of Native PAGE
Native or Non-Denaturing
1. Continuous System: Both gels and
electrophoresis tank have same buffer
composition (Single phase gel; a resolving
gel)….e.g DNAAgarose gel electrophoresis
2. Discontinuous System: Both tank and gels have
different buffers (two phase gel; a stacking gel
and separating or resolving gel) e.g. Polyacryl
amide gel electrophoresis (PAGE) for proteins

17. SDS - PAGE
Page 17
• It is a modified version of PAGE whereby
Sodium-dodecyl-sulphate (SDS) is used.
• SDS is an amphipathic surfactant.
• It denatures proteins by binding to the protein
chain with its hydrocarbon ‘tail’, exposing
normally buried regions and ‘coating’ the
protein chain with surfactant molecules.
• The polar ‘head’ group of SDS adds an
additional benefit to the use of this
denaturant.

18. Structure of SDS
Page 18
(NaC12H25SO4)
also called sodium lauril sulfate or sodium lauryl sulfate

20. ⚫ Mercaptoethanol will break the disulphide bridges.
⚫ SDS binds strongly to and denatures the protein.
⚫ Each protein is fully denatured and open into rod-
shape with series of negatively charged SDS
molecule on polypeptide chain.
SDS is an
anionic
detergent.
The sample is
first boiled for
5min in buffer
containing
20
• Beta-
Mercaptoethanol
• SDS

21. ⚫On average, One SDS molecule bind for every
two amino acid residue.
⚫Hence original native charge is completely
swamped by the negative charge of SDS
molecule.
⚫Also referred as Discontinuous gel
electrophoresis.
21

22. SDS-PAGE
Page 22
WHY ? ? ?

23. • In their native form, proteins fold into a variety
of shapes, some compact, some elongated.
• The rate of migration of native proteins
through a sieving medium is therefore more a
reflection of their relative compactness, and
less an accurate measure of molecular weight.
• Denaturing the proteins nullifies structural
effects on mobility, allowing separation on a
true charge/mass ratio basis.
• It also separates subunits in multimeric
proteins, allowing analysis of large, complex
aggregates.

24. BEFORE SDS
AFTER SDS

25. Principle
• In denaturing PAGE protein samples are heated with
SDS before electrophoresis so that the charge-density
of all proteins is made roughly equal. Heating in SDS, an
anionic detergent, denatures proteins in the sample and
binds tightly to the uncoiled molecule.
• Usually, a reducing agent such as dithiothreitol (DTT) or
2-mercaptoethanol is also added to cleave protein
disulfide bonds and ensure that no quaternary or
tertiary protein structure remains.
• Consequently, when these samples are
electrophoresed, proteins separate according to mass
alone, with very little effect from compositional
differences.

27. (2S,3S)-1,4-
bis(sulfanyl)butane-2,3-diol
(DTT)

28. Differences
Page 28
Native PAGE
• Separation is based
upon charge, size,
and shape of
macromolecules.
• Useful for separation
and/or purification of
mixture of proteins
• This was the original
mode of
electrophoresis.
SDS PAGE
• Separation is based
upon the molecular
weight of proteins.
• The most common
method for
determining MW of
proteins
• Very useful for
checking purity of
protein samples

29. Two gel system
 PAGE is widely used to analyze the proteins in complex
extracts.
 The system actually consists of two gels - a resolving
(aka running) gel in which proteins are resolved on the
basis of their molecular weights (MWs) and a stacking
gel in which proteins are concentrated prior to entering
the resolving gel.
 Gel matrices are permeated with networks of pores
through which the molecules move. The amount of
resistance that the matrix presents to the movement of a
molecule depends on the diameter of the pore as well as
the size and geometry of the molecule

30. Stacking gel: ordering/arranging and conc the
macromolecule before entering the field of
separation. (4% of acrylamide) pH 6.7
• Purpose is to concentrate protein sample in sharp
band before enters main separating gel.
Running gel: the actual zone of separation of
the particle/molecules based on their mobility.
(15% of acrylamide) pH 8.9
Pore size: routinely used as 3% to 30% which
is of pore size 0.2nm to 0.5nm resp.
30

31. 31

32. Movement of particle
[Cl] > [protein-SDS] > [Glycinate]

33. Chemistry of acrylamide polymerization
• The polyacrylamide gels used to separate proteins are formed
by the chemical polymerization of acrylamide and a cross-
linking reagent, N,N’methylenebisacrylamide.
• Polymerization occurs because of free oxygen radicals that
react with the vinyl groups in acrylamide and bisacrylamide.
• The oxygen radicals are generated from the catalyst,
ammonium persulfate (APS), when it reacts with a second
catalyst, N,N,N’,N’-tetramethylethylenediamine (TEMED).

36. ELECTROPHORESIS
INSTRUMENTATION
Page 36

37. •
•
A typical setup consists of a gel slab sandwiched between
two glass plates, with the ends enclosed in upper and
lower reservoirs of buffer
Samples to be run are loaded in wells at the top of the
gel, in conjunction with tracking dye. An electrical voltage
is applied between the upper and lower reservoirs,
causing the samples to migrate down through the gel.

38. Page 13
Procedure of SDS- PAGE

39. Procedure
Set up Gel Load the Buffer
Load Sample
Page 39

40. Assembling the glass plates:
•Assemble the glass plate on a 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 that the bottom ends of the glass plates are properly
aligned.
•Then place it on the casting stand.
Page 40

41. Casting the gels
Page 41
2. Prepare 10%of resolving gel and 4.5% of stacking gel.
•Prepare the separating gel solution by combining all reagents. Do not
add Ammonium persulfate and TEMED.
•Add APS and TEMED to the monomer solution (just before pouring)
and mix well by swirling gently. Pour the solution till the mark. (It is ok if
you introduce air bubbles, add a layer of isopropanol or distilled water
on top of the gel so as to level the poured gel.)
•Allow the gel to polymerize for 20-30 minutes .
•Prepare stacking gel. Mix all reagents except APS and TEMED. Drain
the isopropanol with strips of filter paper .
•Add APS and TEMED to the monomer solution (just before pouring)
and mix well by swirling gently. (Make sure you keep the comb ready by
the side.)
•Place a comb in the stacking gel sandwich. Allow it to polymerize for 10
minutes.

42. Contents of Gel
Page 42

43. Preparation of samples
3.Mix your protein in the ratio 4:1 with the
sample buffer. Heat your sample by either:
a) Boiling for 5-10 minutes. (works for most proteins)
b) 65°C for 10 minutes.
c) 37°C for 30 minutes.
Page 43

44. Running the gel
•To assemble, take out the gels from the casting frame and clamp
them in the gel apparatus. (Make sure that the short plate always
faces inside and if you have got only one gel to run use the dummy
plate that is available to balance).
•When the plates are secured, place them in the cassette and then
lock it.
•Place them in the gel running tank.
Page 44

45. •
•
Fill the inner chamber of the tank with buffer.(Now it
is easy to remove the comb, since it is lubricated).
Remove the comb CAREFULLY (without breaking the
well).
[Now the gel is ready to load the samples]
Page 45

46. •
•
Rinse the loading tip a few times with distilled water.
(Make sure that all the water is poured out before
loading the samples.)
Insert the loading tip to a few mm from the well bottom
and deliver the samples into the well. Rinse the syringe
with distilled water after loading for a few times .
Page 46

47. • Attach the power supply by putting the lid (Make sure
that the connection is in correct way i.e., black - black
and red - red). Set the voltage up to 180 V and run for
1 hour.(Don't allow the dye front to go out of the gel).
Page 47

48. Staining the gel
•After running, switch off the power supply and take out
the gel plates, remove the gel. Place the gel in the staining
solution for 30 minutes.
Page 48

49. Staining solution
Page 49
•Weigh 0.25g of Coomassie Brilliant Blue R250
in a beaker.
•Add 90 ml methanol : water (1:1 v/v) and
10ml of Glacial acetic acid ,mix properly using
a magnetic stirrer.
•Filter through a Whatman No. 1 filter & store
in bottles.

50. Destaining the Gel
• Destain the gel until the bands are properly seen.
Determine the approximate molecular weight of the
visualised protein bands by comparing them with
the molecular weight ladders(markers).
Page 50

51. Destaining solution
Page 51
•Mix 90 ml methanol: water (1:1 v/v)
and 10ml of Glacial acetic acid using a
magnetic stirrer and store in
appropriate bottles.

52. •
•
•
Staining
Coomassie blue-sensitive to 0.1ug of protein
Silver- sensitive to 0.002ug of protein, based on ppt of silver ions
producing brown stain.
• greater sensitivity, radioactive samples can be used, allowing for
exposure over time to produce images on photographic film, as
seen in the sequencing gel on the right
• To calibrate the relative migrations of molecules of different size, a
marker lane is often added, where samples of known size wPiallge27
migrate to reference positions

53. Page 53

54. Relative Mobility
where,
Z = charge on the molécule
E = Voltage applied
and ,
Rf is measured by: f = frictional resistance
Direction of movement is
determined from Z: -
if Z < 0, then →+
if Z > 0, then →-
if Z = 0, then no movement
Page 54

55. Significance of SDS
Page 55
• SDS is a anionic detergent (soap) that can dissolve
hydrophobic molecules but also has a negative charge
• For uniform distribution of charge per unit area(surface)
(q/A)
• For getting the uniform direction of motion of molecules
• If a cell is incubated with SDS, the membranes will be
dissolved and the proteins will be solubilized by the
detergent

56. Advantages of PAGE
Page 56

57. Applications
Page 57
•
•
•
•
Used for estimation of molecular weight of proteins and
nucleic acids.
Determination of subunit structure of proteins.
Purification of isolated proteins.
Monitoring changes of protein content in body fluids.
a) To identify whether a particular protein is pure or not.
b) Separation of proteins, prior to Western Blot transfer.
c) Species identification.
d) Antigen preparation.
e) To measure genetic diversity

58. Potential problems with Polyacrylamide gels
Page 58
– Under loaded (bands invisible)
– Sloppy loading or to little concentration of protein
– Bent bands
– Tearing
– frowning

59. 1. Over loaded 3. Frowning (run too hot)
4. Bent bands. Tearing
2. Tearing
5. Sloppy loading or
too low conc.
Page 59

60. POLYACRYLAMIDE GEL ELECTROPHORESIS
Page 60

61. References
Page 61
• BIOCHEMISTRY by Donald Voet & Judith Voet, Wiley publications.
• BIOCHEMISTRY by Satyanarayana & Chakrapani.
• Biochemistry by Upadhyay , Upadhyay and Nath.
•
•
•
•
http://amrita.vlab.co.in/?sub=3&brch=186&sim=319&cnt=2
http://www.protocolonline.org/prot/Molecular_Biology/Elec
trophoresis/Polyacrylamide_Gel_Electrophoresis PAGE_/in
dex.html
http://www.sciencedirect.com/science/article/pii/00032697
60900361
http://www.nature.com/nature
physci/journal/v230/n12/abs/physci230092a0.html
https://slideplayer.com/slide/3408337/

62. <div style="width:708px"> <strong
style="display:block;margin:12px 0 4px"><a
href="https://slideplayer.com/slide/3408337/"
title="Protein Gel Electrophoresis 1.Native PAGE
2.Native Gradient PAGE 3.Urea PAGE 4.SDS PAGE
5.SDS Gradient PAGE 6.IEF 7.2D PAGE 8.Western
Blot." target="_blank">Protein Gel Electrophoresis
1.Native PAGE 2.Native Gradient PAGE 3.Urea PAGE
4.SDS PAGE 5.SDS Gradient PAGE 6.IEF 7.2D PAGE
8.Western Blot.</a></strong><iframe
src="https://player.slideplayer.com/12/3408337/"
width="708" height="580" frameborder="0"
marginwidth="0" marginheight="0" scrolling="no"
style="border:1px solid #CCC;border-width:1px 1px
0" allowfullscreen></iframe><div
style="padding:5px 0 12px"></div></div>

63. Page 63