Polyacrylamide Gel
Electrophoresis
Jeff Prischmann
Diagnostic Lab Manager
North Dakota State Seed Department
Polyacrylamide Gels
Polyacrylamide gels are formed by the
polymerization of acrylamide with a
crosslinker (usually N, N’ methylene
bisacrylamide).
Polymerization is initiated by the
introduction of a catalyst.
Polyacrylamide Gels
Components
Acrylamide: 30-40% solution
Bisacrylamide: 2% solution
Ammonium persulfate (APS): 10% solution.
TEMED
Buffer
Water
Polyacrylamide Gel
Electrophoresis
Polyacrylamide Gels
PAGE Methods
Troubleshooting
Applications
Polyacrylamide Gels
Catalysts
Ammonium persulfate with TEMED
(N,N,N’,N’ tetramethylethylene diamine)
FeSO4 with 3 % hydrogen peroxide
Photochemical using riboflavin/TEMED
Polyacrylamide Gels
Other Crosslinkers
DATD (N,N’-diallytartardiamide)
BAC (N,N’-bisacrylylcystamine)
PDA (piperazine diacrylate)
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Polyacrylamide Gels
Polyacrylamide Gels
Composition
T=% total concentration (w/v) of
monomer (acrylamide plus crosslinker)
in the gel.
C=% total monomer represented by
the crosslinker.
Pore size is controlled by changing T
and C.
Source: National Diagnostics
Polyacrylamide Gels
Composition
When T is increased, gel pore size
decreases.
Small gel pores are used to separate
smaller molecules.
When C is decreased, it results in a
more open gel pore structure.
Polyacrylamide Gels
Gel Pore Size
Source: National Diagnostics
Source: National Diagnostics
Polyacrylamide Gels
Polyacrylamide Gels
Composition
5% C (19:1 acrylamide/bis) is generally
accepted for denaturing DNA/RNA
separation.
3.3% C (29:1 acrylamide/bis) is
generally accepted for native DNA/RNA
separation.
2.6% C (37.5:1 acrylamide/bis) is
generally used for SDS-PAGE of
proteins.
Source: National Diagnostics
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Polyacrylamide Gels
Steps in Gel Preparation
Prepare the gel casting cassette (wash
and clean glass, treat with hydrophobic
reagent if necessary, and assemble).
Prepare the gel
Pour the gel into the gel casting
cassette.
Allow a minimum of 2 hours prior to
using the gel.
Polyacrylamide Gels
Gel Preparation-Acrylamide/bisacrylamide
Need to know total gel volume, concentrations
of bisacrylamide and acrylamide stock
solutions, % bisacrylamide (crosslinker)
desired, and % acrylamide desired.
Polyacrylamide Gels
Polyacrylamide Gels
Gel Preparation-Acrylamide/bisacrylamide
For gels with separate acrylamide and
bisacrylamide, calculate amounts needed
separately.
For gels with acrylamide and bisacrylamide
together in the same solution, calculate
amounts needed as a single amount.
Stock acrylamide solutions are either 30% or
40%.
Stock bisacrylamide solutions are usually 2%.
Polyacrylamide Gels
Gel Preparation
Example: For DNA gels using 19:1 or 29:1
acrylamide to bisacrylamide ratios:
V = (X) (V1)/40
V = volume of 40 % needed (ml)
V1= total gel volume (ml)
X = % gel desired
Polyacrylamide Gels
Gel Preparation-other components
Buffer
Water
10% (w/v) Ammonium persulfate (1ml per
100ml gel) or other catalyst
TEMED (0.1ml per 100ml gel) or other catalyst
PPE is required when handling and
preparing gels, stains, and etc. (gloves,
lab coat, goggles, etc.)
Acrylamide is a neurotoxin and needs
to be handled with caution.
Gel stains can be toxic (DNA stains) .
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Polyacrylamide Gels
Gel and stain waste needs to be
disposed of properly.
Separate solid and liquid waste.
Acidic /basic waste needs to be
neutralized (TCA).
Polyacrylamide Gels
Polyacrylamide Gels
Resolving gel only
Stacking gels/resolving gels
Gradient gels (% acrylamide increases
from top of gel to bottom)
Polyacrylamide Gels
Gradient Gel Maker
2 main types of gels
Slab gels
Tube gels
Source: National Diagnostics
As component A empties, the
solution leaving the apparatus
becomes more like component B.
Polyacrylamide Gels
Gel Casting Cassette
Glass plates
Gel spacers
Clamps
Sample combs
Gel Casting Stand
Polyacrylamide Gels
Glass plates can vary in size (10cm
wide up to 50cm; length from 52cm
down to 8cm).
Gel spacers/combs can vary in
thickness (1.0mm to 2.0mm).
Sample combs vary in the number of
wells and size of wells.
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Polyacrylamide Gels
Polyacrylamide Gels
Polyacrylamide Gels
Polyacrylamide Gels
Pre-made polyacrylamide slab gels are
available from a number of
manufacturers in different sizes or
concentrations.
Polyacrylamide Gels
Polyacrylamide Gels
Running Gels
Appropriate amount of sample is
added to sample wells.
Tracking dyes used to monitor
progress.
Constant voltage typically used during
electrophoresis.
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Polyacrylamide Gels
Polyacrylamide Gels
Polyacrylamide Gels
Polyacrylamide Gels
Source: Bio-Rad
PAGE Methods
Main Steps
Prepare samples
Prepare gel and buffers
Load samples onto gel
Run gel
Stain gel
Interpret/analysis of gel
Archive (photograph, dry gel)
PAGE Methods
Denaturing PAGE (SDS-PAGE)
Native PAGE of seed protein (wheat
and oat).
Native PAGE of nucleic acids (barley).
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PAGE Methods
PAGE Methods
SDS-PAGE (Laemmli method)
SDS-PAGE
Most commonly used type of PAGE
for protein separation.
Denaturing, discontinuous system
(stacking/resolving gels).
Samples separated by size
Sharp band separation
Source: National Diagnostics
SDS-PAGE uses a stacking gel and
resolving gel combination.
PAGE Methods
PAGE Methods
SDS-PAGE
SDS-PAGE
Source: National Diagnostics
Proteins are stacked between
chlorine ions and glycine .
Source: National Diagnostics
Proteins are separated in the
separation/resolving gel.
PAGE Methods
PAGE Methods
SDS Gel Electrophoresis
Native PAGE (wheat seed protein)
Source: Abcam
ISTA standard reference acid PAGE
method for the separation of
gliadins.
10% acrylamide gels, pH 3.2
Gliadin proteins extracted from
seed using 2-chloroethanol (single
seed or bulk seed).
Buffer components: Acetic Acid and
glycine.
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PAGE Methods
PAGE Methods
Native PAGE (wheat seed protein)
Native PAGE-Wheat
Methyl green used as a tracking dye
in the sample.
Gels run 5 hr at 500V, 20C.
Gels stained with coomassie blue G250.
Visualization in approx 2hr.
PAGE Methods
PAGE Methods
Native PAGE-Wheat
Native PAGE-Wheat
PAGE Methods
PAGE Methods
Native PAGE (oat seed protein)
Native PAGE (oat seed protein)
ISTA standard reference acid PAGE
method for the separation of
gliadins.
10% acrylamide gels, pH 3.2
Avenin proteins extracted from oat
seed using 2-chloroethanol (single
seed or bulk seed).
Methyl green used as a tracking dye
in the sample.
Gels run 2 hr at 500V, 20C.
Gels stained with coomassie blue G250.
Visualization in approx 2hr.
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PAGE Methods
PAGE Methods
Native PAGE-Oat
Native PAGE-Barley DNA
Barley genomic DNA isolated from
leaf/seed.
Barley PCR products
6% acrylamide gels
1.5 hr at 275V
Buffer: TBE
PAGE Methods
PAGE Methods
Native PAGE-Barley DNA
Native PAGE-Barley DNA
Gels stained with SYBR gold or
ethidium bromide.
Visualize in 15-30 minutes
PAGE Methods
PAGE Methods
Native PAGE-Barley DNA
Native PAGE-Barley DNA
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Results
Results
Variety ID or genetic purity tests.
Tests conducted with known standards.
Most varieties have unique banding
patterns or ‘fingerprints’.
Gels photographed, archived, dried.
Troubleshooting PAGE
‘Smiling’ gel front (center of gel running faster
than either end; heat problem, buffer problem).
Vertical streaking of protein (sample overload;
dilute samples, reduce voltage).
Sample does not settle into well bottom (sample
density problem).
Dye front does not migrate (buffer problem,
power supply problem).
Skewed of distorted bands (poor polymerization
around sample wells).
References
Cooke, R.J. 1995. Gel Electrophoresis for the
Identification of Plant Varieties. J.
Chromatography 698:281-299.
C.W. Wrigley. 1995. Identification of Food
Grain Varieties. Chapter 4. Variety
Identification by Electrophoretic Analysis.
Cooke, R.J. 1992. ISTA Handbook of Variety
Testing.
PAGE Advantages
Allows pore sizes to be altered easily.
Superior resolution with small pore size (a
0.1% difference in size can be detected, i.e. 1
base in a 1 Kb DNA fragment)
Virtually no batch to batch differences in
acrylamide or bisacrylamide.
References
Sambrook, J. 2001 Gel Electrophoresis of DNA
and Pulsed-field Agarose Gel Electrophoresis.
Chapter 5 in Molecular Cloning: A Laboratory
Manual. 3rd Editon.
National Diagnostics
(www.nationaldiagnostics.com)
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Questions
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