In an applied electrical field, the SDS-treated proteins will move toward the cathode at different rates
depending on their molecular weight. These different mobilities will be exaggerated by the high-friction
environment of a gel matrix. The gel matrix used for SDS-PAGE is polyacrylamide, which is a good choice
because it is chemically inert and, crucially, can easily be made up at a variety concentrations to
produce different pore sizes giving a variety of separating conditions that can be changed depending on
The electrophoretic apparatus was disassembled and the glass plates were first washed with water and
afterwards they were cleansed with an alcohol and a soft tissue. Then, the resolving gel was prepared by
mixing approximately 4.0 mL of water, 3.3 mL acrylamide solution, 2.5 mL 0.5 M Tris-HCL, 1.0 mL of 10%
SDS solution, 0.06 mL of 10% ammonium persulfate and 0.004 mL of TEMED in a regular test tube. A
micropipette was used in order to transfer the small volume of the various reagent mentioned.
Subsequently, a micropipette was also utilized to fill the plates with the prepared resolving gel. An
ample amount of ethanol was poured on top of the gel to prevent oxygen from diffusing and to promote
polymerization. The mixture was allowed to stand for few minutes until the gel hardened.
Once the resolving gel polymerized, the stacking gel was made. The preparation for the stacking gel was
similar to that of the resolving gel but the volumes of the components differ. The stacking gel was
prepared by combining 2.8 mL of water, 0.83 mL acrylamide solution, 1.26 mL 0.5 M Tris-HCL, 0.05 mL of
10% SDS solution, 0.05 mL of 10% ammonium persulfate and 0.005 mL of TEMED. The comb which is
part of the electrophoretic apparatus was placed in the stacking gel in order to create the wells needed
for the loading of the sample. Again, it was allowed to stand until the gel polymerized.
During the experiment, it was observed that when water, acrylamide solution, Tris-HCL, 10% SDS
solution, 10% ammonium persulfate and TEMED were combined the liquid solution hardens after
several minutes and produces the resolving gel.
The hardening of the solution happens due to the polymerization of the acrylamide and bisacrylamide
molecules in the acrylamide solution.
Acrylamide polymerizes spontaneously in the absence of oxygen, so the polymerization process involves
complete removal of oxygen from the solution. The use of ammonium persulfate and TEMED in the
preparation of the gel initiates the polymerization of acrylamide. TEMED and Ammonium persulfate
serves as a source of free-radical and stabilizer for the polymerization reaction, thus, it follows a freeradical
reaction . The free radicals transfer electrons to the acrylamide/bisacrylamide monomers,
radicalizing them and causing them to react with each other to form the polyacrylamide chain. In the
absence of bis-acrylamide, the acrylamide would polymerise into long strands, not a porous gel. But as
the diagram shows, bis-acrylamide cross-links the acrylamide chains and this is what gives rise to the
formation of the porous gel matrix. The amount of crosslinking, and therefore the pore size and
consequent separation properties of the gel can be controlled by varying the ratio of acrylamide to bisacrylamide.
Tris-HCl is used as buffer for the gel. Addition of Tris-HCl makes the pH of the gel low making the
proteins to release lose a lot of their charge and decrease their mobility .
Glycine can exist in three different charge states, positive, neutral or negative depending on the pH.
Control of the charge state of the glycine by the different buffers is the key to the whole stacking gel
When the power is turned on, the negatively-charged glycine ions in the pH 8.3 electrode buffer are
forced to enter the stacking gel, where the pH is 6.8. In this environment glycine switches predominantly
to the zwitterionic (neutrally charged) state. This loss of charge causes them to move very slowly in the
electric field. The Cl- ions (from Tris-HCl) on the other hand, move much more quickly in the electric field
and they form an ion front that migrates ahead of the glycine. The separation of Cl- from the Tris
counter-ion (which is now moving towards the cathode) creates a narrow zone with a steep voltage
gradient that pulls the glycine along behind it, resulting in two narrowly separated fronts of migrating
ions; the highly mobile Cl- front, followed by the slower, mostly neutral glycine front. All of the proteins
in the gel sample have an electrophoretic mobility that is intermediate between the extreme of the
mobility of the glycine and Cl-so when the two fronts sweep through the sample well the proteins are
concentrated into the narrow zone between the Cl- and glycine fronts.
This procession carries on until it hits the running gel, where the pH switches to 8.8. At this pH the
glycine molecules are mostly negatively charged and can migrate much faster than the proteins. So the
glycine front accelerates past the proteins. The result is that the proteins are dumped in a very narrow
band at the interface of the stacking and running gels and since the running gel has an increased
acrylamide concentration, which slows the the movement of the proteins according to their size, the
separation begins. So the stacking gel ensures that all of the proteins arrive at the running gel at the
same time so proteins of the same molecular weight will migrate as tight bands.
Moreover, the electrophoretic method makes use of the detergent sodium dodecyl sulfate (SDS). SDS binds to the most proteins in amounts roughly proportional to the molecular weight, about one
molecule per two amino acid residues . The bound SDS contributes a large net negative charge,
rendering the intrinsic charge of the protein insignificant and conferring on each protein similar mass-to-charge ratio . In adition, SDS binding partially unfolds protein and helps in breaking it down as it flows
down the gel .
The resolving gel serves as the environment for the proteins as they move at different rates due to
potential difference. Moreover it acts as molecular sieve for the protein, slowing the migration of the
SDS breaks up the two- and three-dimensional structure of the proteins by adding negative charge to
the amino acids. Since like charges repel, the proteins are more-or-less straightened out, immediately
rendering them functionless . Some quaternary structure may remain due to disulfide bonding
(covalent) and due to covalent and noncovalent linkages to other types molecules.
In the methodology, ethanol was poured to the gel. Its main purpose is to make the gel smooth. Ethanol
holds very little water in solution, forming a neat layer on top. Water would make an effective overlay
but would mix with the acrylamide solution, diluting it. In fact, the butanol used is saturated with water
so that it does not dry out the gel mix .
The stacking gel has a low concentration of acrylamide and the running gel a higher concentration
capable of retarding the movement of the proteins. This is because the stacker is simply designed to
perform as a matrix through which samples will pass as they are caught up between moving boundaries
. It is not designed for uniform separation of proteins. Sufficient amount of acrylamide is enough in
order to permit stacking of very large proteins and still retain mechanical strength to make good sample
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