Polyacrylamide Gel

Electrophoresis (SDS- PAGE)

List of Experiments

1. Isolation and purification of genomic DNA from bacteria using BioLit

Genomic DNA extraction Kit
2. Isolation and purification of plasmid DNA from bacteria using BioLit
plasmid DNA extraction kit
3. Analysis of DNA (Genomic and Plasmid) using Agarose gel
electrophoresis
4. Estimation of concentration of unknown nucleic acid(DNA/RNA) and
protein sample using spectrophotometric techniques
5. Analysis of proteins by polyacrylamide gel electrophoresis (SDS- PAGE)
6. To estimate concentration of RNA by Orcinol Method
 SDS PAGE
• Web link
https://vlab.amrita.edu/?sub=3&brch=186&sim=319&cnt=306
 Electrophoresis
• Analytical method used to separate components of a protein mixture
based on their size.
• Migration of a charged particle under the influence of an electric field.
• Biological molecules possess ionisable groups at any given pH
• Exist in solution as electrically charged species either as cations or anions
• Under the influence of an electric field these charged particles will migrate
• Either to cathode or to anode
• Mobility of a substance in the gel depends on both charge and size
• Biological samples needs to be treated -
• Acquire uniform charge
• Electrophoretic mobility depends primarily on size
 SDS
• Different protein molecules with different shapes
and sizes are denatured with SDS so that the
proteins loose their secondary, tertiary or
quaternary structure
• Proteins covered by SDS are negatively charged
• Migrate towards the anode (positively charged
electrode) when loaded onto a gel and placed in
an electric field
• Separated by a molecular sieving effect based on
size
 Principle behind separation
• Separation of charged molecules in an electric field
• Based on the relative mobility of charged species which is related to frictional
resistance.
• Charge of the species:
• Charged molecule migrate towards opposite positively charged electrode
through highly cross linked matrix
• Separation occurs due to different rates of migration
• Occurs by magnitude of charge and frictional resistance related to size
 Polymerization of Acrylamide
• Acrylamide
• Forms linear polymers
• Bisacrylamide
• Cross links two acrylamide molecules
• Polymerized acrylamide (polyacrylamide) forms a mesh-like
matrix suitable for the separation of proteins of typical size.
• More numbers of cross links means smaller
pore size.
• Ratio of Acrylamide and Bisacrylamide determines pore size.
• Ammonium persulfate (APS)- Source of free radicals and is
often used as an initiator for gel formation
• TEMED (N, N, N′, N′-tetramethylethylenediamine) - Stabilizes
free radicals and improves polymerization
• After running the gel
• Determine the relative migration distance (Rf) of the protein standards
and the unknown protein
• Rf = Migration distance of the protein
Migration distance of the dye front
• Rf = ZE
F
• Z = charge on the molecule
• E = Voltage applied
• f = frictional resistance
Discontinuous buffer system
• Stacking Gel
• Resolving Gel

• Utilizes differences in ionic strength and pH between the electrophoresis

buffer and the stacking gel buffer
• Involves a phenomenon known as isotachophoresis
• Technique in analytical chemistry used for selective separation and concentration of
ionic analytes
• Charged analytes are separated based on ionic mobility, a quantity which tells how
fast an ion migrates through an electric field.
Running Buffer

Stacking Gel

Resolving Gel
 Stacking gel
• Very large pore size (4% acrylamide)
• Allows the proteins to move freely and concentrate, or stack, under the effect of
the electric field.
• Result three species of interest adjust their concentrations
• [Cl-] > [protein–SDS] > [glycinate]
 Leading to stacking of the protein sample
• Stacking occurs by differential migration of ionic species which carry the electric
current through the gel.
• On application of electric field
• Negatively charged molecules start migrating to the positively charged electrode
• Cl- ions, having the highest charge/mass ratio move faster, being depleted and concentrated
at anode end.
• SDS coated proteins has a higher charge/mass ratio than glycine so it moves fast, but slower
than Cl-.
• When protein encounters resolving gel it slows the migration because of increased frictional
resistance, allowing the protein to stack in the gel.
Resolving Gel Interactions

• Glycine upon reaching resolving gel becomes negatively charged and

migrates much faster than protein due to higher charge/mass ratio.
• Now proteins are the main carrier of current and separate according
to their molecular mass by sieving effect of pores in gel
Source: Science made easy
 Materials Required For PAGE
 Acrylamide solutions (for resolving & stacking gels)
Isopropanol /distilled water
Gel loading buffer
Running buffer
Staining, destaining solutions
Protein samples
Molecular weight markers

• 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
• 30% Polyacrylamide solution –
• 29g acrylamide+ 1g bisacrylamide in 50 mL of water, dissolve completely using a
magnetic stirrer, make the volume upto 100mL.
• Keep the solution away from sunlight.
• 1.5 M Tris, pH 8.8
• 1 M Tris, pH 6.8
• 10% SDS
• (10 g SDS in 100mL distilled water)
• 10% ammonium persulfate
• (0.1 g in 1 ml water). It should be freshly prepared.
• 1x SDS running buffer( pH ~8.8)
Resolving Gel (10 %) Volume Stacking gel (5%) Volume
dH2O 4 ml
dH20 5.65 ml
30 %Acrylamide 3.3ml
30% acrylamide mix 1.65 ml
1.5M Tris pH 8.8 2.5 ml
1.0M Tris pH 6.8 2.5 ml
10% SDS 0.1 ml
10% SDS 0.1 ml
10% Amoonium per sulphate 0.1 ml 10% ammonium persulfate 0.1 ml
TEMED 0.004ml TEMED 0.004 ml
• Staining Solution

• (50:10:40 /Methanol: Acetic acid: H2O) + Commassie Brilliant Blue R-250

• Commassie Brilliant Blue R-250
• Methanol
• Acetic Acid Solution
• Water
Stain for an hour in staining solution

• Destaining Solution
• (50:10:40 /Methanol: Acetic acid: H2O)
• Gel loading buffer:
• To make 10 mL of 4X stock:
1M Tris-HCl pH 6.8. - 2.0 ml
SDS - 0.8 g
100% glycerol - 4.0 ml
14.7 M β-mercaptoethanol - 0.4 ml
0.5 M EDTA - 1.0 ml
Bromophenol Blue - 8 mg
 Gel Loading Buffer
• β-mercaptoethanol - reduces any disulphide bridges present that are holding together the
protein tertiary structure
• SDS -
• Binds strongly to, and denatures, the protein.
• Each protein in the mixture fully denatured
• Opens up into a rod-shaped structure with a series of negatively charged SDS molecules along the polypeptide
chain.
• Original native charge on the molecule is therefore completely swamped by the negatively
charged SDS molecules.
• Bromophenol blue –
• Ionisable tracking dye
• Allows the electrophoretic run to be monitored
• Sucrose or glycerol –
• Gives the sample solution density
• Allowing the sample to settle easily through the electrophoresis buffer to the bottom when injected into the
loading well.
Source: Bio Rad
OBSERVATION: KDa
80
66
56
50

43

35

20

14

12% SDS PAGE

Procedure
1) Assemble glass plates, properly cleaned with a help of detergent and dried.
2) Check for any kind of leakage.
3) Clamp the entire set on a gel casting stand
4) Prepare 12% separating gel in a 50 ml beaker as in materials.
5) Add APS and TEMED and swirl the beaker to ensure proper mixing
6) Pour the separating gel in between the glass plates.
7) Overlay the gel with butanol or water.
8) Allow the gel to polymerize for 20 min and do not disturb.
9) Once the gel has polymerized, drain the water and wipe any sticking water with a
filter paper strip.
10) Prepare stacking gel in a 10 ml beaker as in Materials
Procedure
11) Add TEMED and APS. Gently swirl
12) Pour the stacking above the separating upto a height of 1.5 cm.
13) Quickly place the comb without formation of any air bubble.
14) Allow polymerization for 20 min.
15) After polymerization, remove the plate and place in the tank filled with running buffer.
16) Remove the comb
17) Load the samples using a pipette.
18) Load 5µl of protein sample and 20 µl of marker as provided in SRL Biolit SDS PAGE
teaching kit.
19) Run the gel at constant voltage of 50V till the dye front reaches the bottom of the gel.
20) Remove the gel very carefully and place in a staining try for 1 h.
21) Destain for 1 h and observe migration of the bands.
Precautions

• 1) Loading of samples should be done carefully to avoid inter well

mixing and avoid puncturing the well.
• 2) Care should be taken at the time of opening of plates to avoid
damage to glass plates.
• 3) Gel is fragile and should be handled with care.
• Gloves should be worn, while performing SDS-PAGE.
• To ensure proper alignment, all the requirements should be clean.
• Special attention should be paid while using acrylamide(since it is a
neurotoxin)