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the presence of a constant potential difference such that the proteins of various sizes
flow through it at diverse speeds and hence one can tell the components of the solution
established on the positions of a dye stain which binds to the proteins. The principle of
the technique is that the mobility of a charged molecule is directly proportional to its net
charge and inversely proportional to the resistance of the solution through which it is
moving. The mobility is also proportional to the voltage of the field. All molecules
experience the same voltage in the gel. Thus, mobility is governed by net charge and
resistance.
sulfates, mainly the lauryl; lowers surface tension of aqueous solutions; used as fat
determine the relative abundance of major proteins in a sample, and to determine the
distribution of proteins among fractions. The purity of protein samples can be assessed
staining methods can be used to detect rare proteins and to learn something about their
scarce gene products, to find similarities among them, and to detect and separate
There are two kinds of matrix. The Polyacrylamide or agarose which are substances
that gel can be made from. Agarose are complex sugar chain from red seaweed. It has
a large pore size which is good for separating large molecules quickly. On the other
hand, Polyacrylamide is a chain of acrylamide molecules that has a small pore size
good for separating small molecules. As it was said previously, mobility is administered
by net charge and resistance. Resistance provided by the matrix is in relation to the
molecular size and shape of the molecules moving through it. For a given pore size
which is the size of the holes in the matrix through which the molecules move, bulkier
molecules experience greater resistance and therefore move more slowly through the
matrix than petite molecules. The longer the DNA molecule, the longer the time it takes
to slip through the gel. Electric charge also plays a role in the separation. Molecules
with negative charge will be attracted to the positively charged node while molecules
When a mixture of proteins is dragged by the electric field into the gel, they begin to
separate on the basis of size. But the smallest of the proteins will have started to make
their way through the gel even before all the sample has entered the gel. Thus smaller
sized molecule move faster. Through the stacking gel, the entire sample of 10 or 20 µl
condense into the narrowest possible band right at the interface of the gel, lining them
up at the starting line. The Polyacrylamide gels are run in a vertical orientation, unlike
agarose gels, which are horizontal; and that SDS-coated protein are negatively
The polyacrylamide gel is created in two layers, the larger, running gel on the bottom,
and a narrow stacking gel on top. The reservoirs on the top and the bottom of the gel
contain a buffer made with glycine and adjusted to pH 8.3. The stacking gel has a pH of
6.8, while the resolving gel has a pH of 8.8. After you load the samples into the wells at
the top of the gel, you will turn on the voltage, and current will flow through the gel from
one reservoir to the other, carried by negatively charged molecules. The current in the
top reservoir is carried by some of the glycine molecules; but note that at pH 8.3, only
Proteins first enter at the top surface of the stacking gel. Glycine at pH 6.8 has almost
no negative charge, so here the current is carried mainly by Cl- in the stacking buffer.
Because of their small size the Cl- ions rush ahead of the proteins, which move more
slowly. This results in a concentration gradient of Cl- ions, which pile up towards the
bottom of the stacking gel. The effect is that within the stacking gel, the SDS-coated
protein molecules are sandwiched between glycine molecules above and Cl below, and
are compressed into a band of much smaller volume than was originally loaded. As the
current continues to flow, the proteins move into the resolving gel. But glycine
molecules in the resolving gel buffer at pH 8.8 become negatively charged and move
quite fast, nearly keeping up with Cl- ions, and leaving the proteins trailing behind. It is
at this point, when the proteins enter the resolving gel, that they are carrying the current
in the trail of the Cl- and glycine anions, and their mobility is a function of molecular size
alone.
The purpose of the SDS and the heat is for the Sodium Dodecyl Sulfate when heated
with the protein binds to the amino acids in the protein and hence destabilizes the
hydrogen-bonding which renders the protein its structure. This “denatures” the protein
by making all of them more or less cylindrical in shape. This ensures that the proteins’s
mobility which regulates their stacking sequence depends less on the structure and
more on the molecular mass. Also, anions of SDS bind to the main peptide chain at a
ratio of one SDS anion for every two amino acid residues. This effectively imparts a
negative charge on the protein that is proportional to the mass of that protein (about
1.4gSDS/gprotein). This negative charging is essential for the migration of the proteins
(precisely the proteinSDS complex) towards the anode during electrophoresis. Figure 1
protein solution mainly to break the di-sulfide bonds in the protein which also help keep
cleaves the disulfide bond I proteins. This is important ecause disulfide bonds, which
can exist within or between polypeptide chains, prevent polypeptide from fully unfolding.
Figure 2 reaction by β−Mercaptoethanol
Polymerization steps happened are first the decomposition of the persulfate ion to give
a free radical catalyzed by the TEMED reagent.Figure 3 shows the reaction. The
The Glycine provides the ion with the slowest mobility which helps in stacking the
proteins during the transport in the presence of the electric field. The Chloride ions
provide the background conducting medium and is the “leading ion” being the ion
around with the highest mobility. Hence the proteins being of intermediate mobility get
stacked between the chloride and the glycine ions. There are different pH’s because
glycine can exist in three different charged states, positive, neutral or negative
depending on the pH. The charged state of the glycine can be controlled by different
buffers. The Tris-HCl buffer was used to conduct the current from the cathode to the
While the Coomassie Blue R-250 is an anionic dye used in the staining solution. This
binds with proteins non-specifically. As SDS is also anionic, it may interfere with staining
The stacking gel works through a process in which when the power is turned on, the
negatively charged glycine ions in the pH 8.3 electrode buffer are required to enter the
stacking gel, where the pH is 6.8. Glycine is thus in the neutral state. They move very
The Cl- ions which came from the Tris-HCl moves faster and forms an ion front that
migrates ahead of the glycine. The separation of Cl- ions from the Tri counter ion
creates a narrow zone with a steep voltage gradients that pulls the glycine along behind
it. This results to a two narrowly separated fronts of migrating ions namely the Cl and
Experimental methodology
Half of the culture (7.5mL) was transferred to a 15mL centrifuge tube. It was
centrifuge for 3 minutes at 12,100 rpm. The supernatant was carefully removed
and the cell was washed three times with distilled water. The tube was vortexed
added and mixed and then was transferred to Eppendorf tube. The proteins were
B. SDS-PAGE
The glass plates and comb were cleaned with detergent and water. The glass
plates and comb were rinsed with ethanol and dried. The glass plates were
assembled on the casting tray. The comb was inserted between the glass plates
and the end of the comb was marked with a marking pen. The running or
separation gel were one cm below the end of the comb. The comb was removed
and the separating gel was prepared. Gloves were worn during the preparation of
running and stacking gel because 30% acrylamide/ 0.8% bis acrylamide is
neurotoxin.
Polymerization will start as soon as TEMED was added. With the help of P-1000
the solution was gently swirled and poured into the glass plates until the mark
made on the glass plate previously. The solution was slowly covered with water
to even out the edges and to prevent oxygen from diffusing into the gel. Ten to
twenty minutes were waited until the remaining gel in the beaker was
polymerized. Five percent stacking gel was prepared in a small beaker by mixing
the solutions in the correct order below. While preparing the staking gel, the
water was removed from the separating gel by tilting and rinsing with distilled
water. The fluid was drained as much as possible using paper towel.
The mixture was swirled and the stacking gel was added on top of the separating
gel. The comb was inserted in the stacking gel. The gel was allowed to
polymerize for about 5 to 10 minutes. After stacking gel has polymerized, the
comb was removed and washed with the running buffer to remove the
unpolymerized acrylamide. The gel was attached to the vertical gel apparatus.
Enough buffer was added until the well were completely filled with the buffer.
weight standards were prepared for loading by heating them in a boiling water
bath for 3 min. and then was kept on ice until they are loaded onto the gel.
predetermined order were loaded into the wells. An equal volume of IX sample
buffer was loaded into wells that are unused. The power lid was attached to the
apparatus and the gel was run at 20V until the proteins are well into the stacking
gel, then 100V until the tracking dye reaches the bottom of the gel. The power
was turned off and the wires were unplug from the power supply. The glass plate
was removed and using a spatula, the plates were pried apart making sure the
gel is not torn apart. The orientation of the gel was marked by cutting off the left
The gel was fixed in fixing solution for 1 hr to overnight with gentile agitation. The
solution was discarded when done. The gel was stained with enough volume of
Coommassie Blue Stain for 1 hour at room temperature. The staining solution
was saved. The gel was destained in enough volume of destaining solution for 30
minutes at room temperature. The destain step was repeated several times. The
References