Poly-acrylamide Gel Electrophoresis

Poly-acrylamide
Gel
Electrophoresis

Mohit Kumar Ram and
Jitendra Kumar
CoF Mangalore

jitenderanduat@gmail.com

Gel
• Substantially dilute cross-linked system, which exhibits
no flow when in the steady-state
• Solid, jelly-like material that can have properties ranging
from soft and weak to hard and tough
• By weight, gels are mostly liquid, yet they behave like
solids due to a three-dimensional cross-linked network
within the liquid
• ExamplesPolyacrylamide gel, Silica gel, Starch gel, Agarose gel etc.

Types of Gels
• Organogels
• Xerogels
• Hydrogels


Organogels
• An organogel is a non-crystalline, non-glassy thermoreversible
(thermoplastic) solid material composed of a liquid organic phase
entrapped in a three-dimensionally cross-linked network
• The solubility and particle dimensions of the structurant are
important characteristics for the elastic properties and firmness of
the organogel
• Organogels have potential for use in a number of applications, such
as in pharmaceuticals, cosmetics, art conservation, and food
• Examplesorganic solvent, mineral oil, or vegetable oil

Xerogels
• A xerogel is a solid formed from a gel by drying without
shrinkage
• Xerogels usually retain high porosity (25%) and enormous
surface area (150–900 m2/g), along with very small pore size
(1-10 nm)
• When solvent removal occurs under hypercritical (supercritical)
conditions, the network does not shrink and a highly porous,
low-density material known as an Xerogel is produced
• ExampleSilica gel


Hydrogels
• Hydrogel (also called aquagel) is a network of polymer chains that are
hydrophilic, sometimes found as a colloidal gel in which water is the
dispersion medium
• Hydrogels also possess a degree of flexibility very similar to natural tissue,
due to their significant water content(99%)
• Hydrogels which are also known as „Smart Gels‟ or „Intelligent Gels‟
• These hydrogels have the ability to sense changes of pH, temperature, or the
concentration of metabolite
• It is common used gel in laboratories
• ExamplesStarch gel, Agarose gel, jitenderanduat@gmail.com gel etc.
Polyacrylamide

Acrylamide
• Acrylamide (or acrylic amide) is a chemical compound with the chemical
formula C3H5NO
• It is a white odorless crystalline solid, soluble in water, ethanol, ether,
and chloroform
• Acrylamide is prepared on an industrial scale by the hydrolysis
of acrylonitrile by nitrile hydratase
• It is carcinogenic as well as Neurotoxic compounds
• Most acrylamide is used to synthesize polyacrylamides polymeririsation
process
• It is used in the manufacture of dyes, Waste water treatment and
other monomers

Polyacrylamide
• 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.

Polyacrylamide gel
• It is a white odorless gel, soluble in water
• After polymerization of acrylamide it get cross-linked
structure

• TEMED stabilizes
polymerization

free

radicals

and

improves

• Here, the toxic affect of acrylamide get vanish (95%)
• Amount of polyacrylamide salt dissolved (conc.) is directly
proportion to cross –linked nature of gel

Polyacrylamide gel Preparation
• Polyacrylamide gels are prepared by the free radical
polymerization of acrylamide and the cross linking agent
N N‟ methylene bis-acrylamide
Acrylamide + N N’ methylene bis acrylamide
Ammonium persulfate (catalyst)
Chemical Polymerization

+
TEMED
(N,N N’ N’ tetramethylethylene diamine)

Polyacrylamide


Electrophoresis
• Electrophoresis, also called cataphoresis, is the motion of dispersed
particles relative to a fluid under the influence of a spatially
uniform electric field
• This electrokinetic phenomenon was observed for the first time in
1807 by Reuss

• Electrophoretic mobility μe defined as:
• Examples- DNA electrophoresis
- Gel electrophoresis (SDS-PAGE)
- Pulsed field gel electrophoresis( technique used for the
separation of large deoxyribonucleic acid (DNA) molecules by
applying an electric field) etc.

Poly-Acrylamide Gel Electrophoresis(PAGE)
• Electrophoresis in which we use polyacrylamide gel as a
sieving/filtering material
• Poly-Acrylamide Gel Electrophoresis (PAGE) is used for
Qualitative Characterization of protein
• This procedure is limited to the analysis of protein with a weight
range of 14,000-100,000 Da
• It is possible to extend the weight range of an electrophoresis gel
by various techniques (gradient gel or particular buffer system)

Sodium Dodecyl Sulphate Poly-Acrylamide
Gel Electrophoresis(SDS-PAGE)
 It is a type of Poly-Acrylamide Gel Electrophoresis in
which, preliminary process is done with help of SDS
 The equipment and supplies necessary for conducting
SDS-PAGE includes:
• An electrophoresis chamber and power supply
• Glass plates(a short and a top plate)
• Casting frame
• Casting stand
• Combs

Structure of SDS


Significance of SDS
• SDS (sodium dodecyl sulfate) 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 soluablized by the
detergent

Action of SDS


Procedure of SDS-PAGE


Preparing of Sample
• Mix your protein 4:1 with the sample buffer. Heat your
sample by either:
a) Boiling for 5-10 minutes (Works for most proteins)

b) 65ºC for 10 minutes (If you have smearing using the
above procedure)
c) 7ºC for 30 minutes (Membrane proteins or others that
do not enter the gel otherwise may benefit from this type
of sample preparation)

Take sample from any part of fish
Each sample must contain 50 µg of protein
(For example, if you calculated that your protein yield was 5 mg protein /mL (5
µg/µL), you would need 10 µL of that fraction)

Place the appropriate volume (based on protein concentration) of each
sample into a labeled microfuge tube

Add 1/4 volume of 4x Sample Buffer to each sample

Place the microfuge tubes containing your sample and sample buffer in a
boiling water bath and boil the samples for 2 minutes
(Remove the microfuge tubes and place tubes on ice. Chilling the samples keeps them
dense so that they “sink” when placed in the wells)

Insert the precast gel to the gel apparatus
Add 1x Running Buffer to the buffer chambers of the electrophoresis apparatus
Connect the leads to a power supply and electrophorese the samples until the
bromophenol blue dye front has traveled to the very bottom of the gel (200
V for ~ 45 minutes)
After electrophoresis, carefully remove the fragile gel from between the glass
plates, and submerge the gel in Coomassie Blue stain. Shake gently on the
shaker for at least 30 minutes
Remove the gel from the stain solution and place in Destain I for 15 minutes to
1hr. Remove and put in Destain II 1 - 4 hours until the background is clear
Put your destained gel on a piece of saran wrap or in Ziploc bag and
photograph it with a digital camera

Chemical Preparation
Coomassie Blue Stain
• 2.5 g Coomassie Brilliant Blue R, 440 mL methanol,
480 mL water, 80 mL glacial acetic acid, filter before
use

5x Electrode (Running) Buffer
• 45 g Tris-base (15 g/L)
216 g glycine (72 g/L)
15 g SDS (5 g/L)

4x Sample Buffer
• 4.0 mL distilled water
• 1.0 mL of 0.5 M Tris-HCl, pH 6.8
• 0.8 mL glycerol
• 1.6 mL of 10% (w/v) SDS
• 0.2 m L of 0.05% (w/v) bromophenol blue
(Store at room temperature. Immediately before use add 0.4
mL B-mercaptoethanol)

Prepare acrylamide gel
• Consist of 30% acrylamide, 0.8% bisacrylamide,
SDS,and a buffer with an adjusted pH
• Store at 4ºC in the dark
• The ratio of acrylamide to bisacrylamide can be
varied for special purposes

Choose a percentage acrylamide based
on the molecular weight range of
proteins you wish to separate
Gel Percentage(%)
Molecular weight
Range

7

10

12

15

50-500kDa

20-300kDa

10-200kDa

3-100kDa

Destaining I solution
(50% methanol, 5% acetic acid, freshly made)
Destaining II solution
(7% acetic acid, 5% methanol, freshly made)

Staining solution
• Dissolve 0.25 g of Coomassie brilliant blue in 45 ml of
methanol. Add 45 ml of H2O and 10 ml of acetic acid.

Stacking Gel Solution (4% Acrylamide)
•
•
•
•
•
•

H2O
0.5 M Tris-HCl, pH 6.8
20% (w/v) SDS
Acrylamide/Bis-acrylamide
(30%/0.8% w/v)
ammonium persulfate (APS)
(10% (w/v)

• TEMED(Tetramethylethylenediamine)

3.075 ml
1.25 ml
0.025 ml
0.67 ml
0.025 ml

0.005 ml

Data analysis
Calculate the Rf (ratio of the fronts) of each protein
standard, using the equation:
Rf = distance of protein migration/distance of dye
front migration.
• Using computer, plot the log of the molecular
weight on the Y axis and the Rf on the X axis

Importance of SDS-PAGE in Modern
Ichthyotaxonomy
• To detecting the various diseases of fishes and
shellfishes on molecular level
• SDS-PAGE denotes technique for identifying genetic
variation in fishes at the molecular level
• Provides a basis to rearrange the species according to
their molecular behavior
• Gives very satisfying and accurate result about the
protein pattern and their types

REFERENCES
• http://www.nature.com/nature
physci/journal/v230/n12/abs/physci230092a0.html
• http://www.sciencedirect.com/science/article/pii/0003269760900361
• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC343768/pdf/nar004780306.pdf
• http://pubs.acs.org/doi/abs/10.1021/ma00178a020
• http://en.wikipedia.org/wiki/Polyacrylamide
• homepages.gac.edu/cellab/chpts/chpt4/ex4-1.html
• http://www.protocolonline.org/prot/Molecular_Biology/Electrophoresi
s/Polyacrylamide_Gel_Electrophoresis__PAGE_/index.html
• http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protoco
lweek11.html
• http://course1.winona.edu/sberg/307/Labs/documents/SDSpage.doc

THANK yoU

Poly-acrylamide Gel Electrophoresis

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Poly-acrylamide Gel Electrophoresis