5/5/2019 Principle and Protocol of Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) – Creative Biomart Blog
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Principle and Protocol of Sodium Dodecyl
Sulphate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE)
Posted By biomart on November 17, 2015
Principle
The concentration of polyacrylamide gels can be prepared as
required in two electrophoresis systems —called “continuous
system” and “discontinuous system”. The biggest feature of
“discontinuous system” lies in its greatly improved sample
separation resolution. Main features of this electrophoresis are: (1)
Use of two gel systems with di erent concentrations; (2) Solution
composition and pH are di erent for the preparation of the two gel
and are also di erent from electrophoresis bu er composition and
pH in electrophoresis tank. In the experiment, electrophoresis gel is
divided into two layers: the upper one is a macroporous gel with low
concentration, called stacking gel, bu er for the formulation of this
layer is Tris-HCl, pH6.7; the lower one is hole glue with high
concentrations, called separating gel or electrophoresis gel , and the
bu er for this is Tris-HCl, pH8.9. Electrode bu er in the
electrophoresis tank is Tris- glycine, pH8.3. Obviously, the gel
concentrations, compositions, pH and the electrophoresis bu er
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5/5/2019 Principle and Protocol of Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) – Creative Biomart Blog
systems are di erent from each other, thus forming a discontinuous
system.
During the experiment,
the protein sample was
loaded in the stacking
gel. To prevent protein
sample di using in the electrode bu er, adding an equal volume of
40% sucrose or 50% glycerol to increase the density would be a
good choice. To observe the mobility of protein samples, it’s better
to add bromophenol blue dye or some other tracer dyes into the
sample. These colored substances can migrate faster than any
macromolecules. As long as the dye dose not move out of the gel,
there would be no danger for the sample.
In the discontinuous system, as soon as the power is turned on,
Glycine, proteins, chloride ions and bromophenol in HCI would be
dissociated into anion, forming an ion ow and moving to the
anode. Its mobility depends on the number of electric charges of the
ion, molecular size and shape. However, when the glycine ions of
electrophoresis bu er (pH8.3) entered into the stacking gel and
encountered lower pH (6.7), which lowered down by nearly two
units, almost close to the isoelectric point (5.97) of glycine, the
dissociation degree of glycine suddenly drop, the amount of charge
reduced signi cantly and then the mobility became slower.Protein
sample entered into the stacking gel, pH changes has impact on its
dissociation degree either, but the impact is much smaller than on
glycine. Thus it has larger mobility than glycine. What’s more, the
pore size of stacking gel is too large to cause obstruction to protein
molecular. So, in the stacking gel, the mobility of various ions is in
the order of: glycine <protein <BPB <Cl.
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5/5/2019 Principle and Protocol of Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) – Creative Biomart Blog
The decline of dissociation degree after the glycine entering into the
stacking gel makes the sudden absence of mobile ions owing,
resulting in reduced conductivity and electric current decline.
However, the entire electric current of the other part of the
electrophoresis system remain unchanged. On the basis that
conductivity is inversely proportional to potential gradient (E=I/n, E
stands for potential gradient, I stands for current intensity and N
stands for conductivity), there suddenly formed a high local
potential gradient between Leading Ion-CI and slow ion-glycine.
Protein components in this local potential gradient region quickly
migrate to the CI-ions region at di erent speed under the function
of the high electric eld. Through this process, the protein sample
has been concentrated for several hundred fold and the protein
components are arranged in a certain order to form layer.
When the ion ow continued to move forward and entered into the
resolving gel prepared by pH8.9 bu er, the protein molecule
encountered resistance. Then the mobility became slow. At the
same time, under the conditions of pH8.9, glycine would fully
dissociate. Its electricity would increase, eliminating the
phenomenon of ion missing. Each section of the gel recovered with
a constant electric strength. Proteins begin to migrate at di erent
rates, because of the sieving properties of the gel. Smaller protein-
SDS complexes migrate more quickly than larger protein SDS
complexes. Within a certain range determined by the porosity of the
gel, the migration rate of a protein in the running gel is inversely
proportional to the logarithm of its MW.
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5/5/2019 Principle and Protocol of Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) – Creative Biomart Blog
Seen from the principle above, main advantages of discontinuous
polyacrylamide gel electrophoresis is that when the protein samples
go through the stacking gel, they can form a tightly compressed
layer and ow into the separating gel. With the protein components
separated previously and compressed into layer, it can reduce the
interference caused by the zone overlapping, thus improving the
distinguish ability of electrophoresis. Just because of this advantage,
a small amount of protein samples can be well separated too.
Materials and Reagents
1. 30% acrylamide: weigh 29g acrylamide, 1g N, N – methylene bis-
acrylamide. Add 60 ml warmed deionized water and heat to 37 ℃.
Add deionized water to make a nal volume of 100ml; lter; Then we
have 30% (w / v) acrylamide stock solution; Acrylamide and bis-
acrylamide were transformed slowly into acrylic acid and double
acrylic acid during storage, so the pH of the solution should be no
more than 7.0 and it should be placed in a brow bottle at 4 ℃.
2. 10% sodium dodecyl sulfate (SDS): weigh 10g SDS and 90ml
deionized water; heat to 68 ℃ and add a few drops of concentrated
hydrochloric acid until the pH becomes 7.2; then water to 100ml;
after the whole processes, we have 10% (w/v) SDS.
3. Stacking gel bu er (1mol / L Tris-HCl pH 6.8): dissolve 12.12g Tris
in 80ml deionized water. Adjust the pH to 6.8 with concentrated
hydrochloric acid; add deionized water to 100ml and store at 4℃.
4. Resolving gel bu er (1.5mol / L Tris-HCl pH 8.8): dissolve 18.16g
Tris in 80ml deionized water; adjust the pH to 8.8 with concentrated
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5/5/2019 Principle and Protocol of Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) – Creative Biomart Blog
hydrochloric acid; add deionized water to 100ml; store at 4 ℃.
5. 10% ammonium persulfate (AP): ammonium persulfate provides
the free radical necessary for the catalysis of the Polymerization of
Acrylamide and Bis-acrylamide; Use deionized water to prepare a
small amount of 10% (w/v) solution and store at 4 ℃. Since
ammonium persulfate will decompose slowly, it should be freshly
prepared every other week.
6. TEMED (N, N, N, N – tetramethylethylenediamine): by catalyzing
ammounium persulfate to form free radicals, TEMED accelerated
the polymerization of acrylamide and bis-acrylamide. Since TEMED
only functions in a free base form, the polymerization reaction
would be inhibited when the pH is low.
7. Tris- glycine electrophoresis bu er: weigh 15.1g Tris and 94g
glycine; Dissolve in 900ml deionized water; then add 50ml 10% (w/v)
SDS and deionized water to 1000ml. Dilute 5-fold when using. The
nal concentration would be: Tris, 25mmol/L; glycine, 250mmol/L;
SDS, 0.1% and the pH of the bu er is 8.3.
8. Polyacrylamide gel electrophoresis tank and electrophoresis
power supply.
9. Transfer pipette and tip, etc.
Operating Method
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5/5/2019 Principle and Protocol of Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) – Creative Biomart Blog
1. Assemble glass plate according to the vertical electrophoresis
tank instructions; determine the concentration and volume of the
separating gel; prepare the desired separating gel according to the
ingredients listed for the preparation of Tris- glycine SDS-
polyacrylamide gel electrophoresis.
2. Inject the separating gel into the gap of the two glass sheets
quickly, leaving space for the infusion of stacking gel (comb teeth
length plus 1cm); cover the separating gel with 0.1% SDS carefully(
when the concentration of acrylamide ≤ 8%) or isobutanol or water
(when the acrylamide
concentration ≥10%); the cover
layer can prevent the di usion of
oxygen into the gel and inhibit
the polymerization of the gel;
place the gel vertically at room
temperature.
3. When the polymerization of the separating gel completed, pour
the cover liquid; wash the top of the gel for several times to remove
the acrylamide that were unpolymerized; exclude the liquid on the
gels as far as possible.
4. Determine the volume of the stacking gel in need; prepare the
desired stacking gel according to the ingredients listed for the
preparation of Tris- glycine SDS-polyacrylamide gel electrophoresis
of stacking gel; Pipette the stacking gel directly on the separating gel,
and insert clean supporting comb immediately, to avoid air bubbles;
then add stacking gel solution to ll the gap between comb. Remove
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5/5/2019 Principle and Protocol of Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) – Creative Biomart Blog
the comb after stacking gel polymerization, then the sample hole is
formed.
5. Dilute 5-fold of the Tris- glycine electrophoresis bu er stock
solution with deionized water; pour the solution into electrophoresis
tank; ll the sample hole so that the bubbles in the sample holes can
be ruled out through the electrophoresis bu er.
Posted in Protocol Tagged Protocol of SDS-PAGE, Resolving gel buffer, SDS-
PAGE, Stacking gel buffer
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