Polyacrylamide gel electrophoresis

Polyacrylamide Gel
Electrophoresis(PAGE)
by Abhi Giri

Acrylamide
• 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

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 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

Gel Types
• 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.
• 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.
Agarose Polyacrylamide Gel

What is 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.

Electrophoresis
• Electrophoresis is a separation technique that is
based on the movement of charged particles in an
electric field.
• The term electrophoresis was coined from a
Greek word “Phoresis” which means “Being
Carried Away”.
• Hence literal meaning of the word electrophoresis
means “to carry with electricity.”
• This electro kinetic phenomenon was observed
for the first time in 1807 by Reuss.

PRINCIPLE
• Any charged ion or molecule
migrates when placed in an electric
field, the rate of migration depend
upon its net charge, size, shape and
the applied electric current.
• Can be represented by following
eq.

ELECTROPHORESIS
INSTRUMENTATION

Poly Acrylamide Gel
Electrophoresis
• 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.

• 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.

Procedure of SDS-
PAGE

Procedure
Set up Gel Load the Buffer
Load Sample

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.

Casting the gels
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.

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.

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.

• 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]

• 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 .

• 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).

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.

Staining solution
•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.

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).

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

• 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 will
migrate to reference positions

• 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

SDS - PAGE
• 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.

Differences
• 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.
• 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
Native PAGE SDS PAGE

• 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.

Relative Mobility
Direction of movement is
determined from Z: -

if Z < 0, then +→

if Z > 0, then -→

if Z = 0, then no movement
Rf is measured by:

where,
Z = charge on the molécule

E = Voltage applied

and ,

f = frictional resistance

Significance of SDS
• 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

Applications
• 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

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

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

POLYACRYLAMIDE GEL ELECTROPHORESIS

References
• 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

Polyacrylamide gel electrophoresis

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