Introduction

Electrophoresis is a technique by which a solution of proteins is passed through a gel in

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.

Sodium dodecyl sulfate is an anionic surfactant, usually a mixture of sodium alkyl

sulfates, mainly the lauryl; lowers surface tension of aqueous solutions; used as fat

emulsifier, wetting agent, detergent in cosmetics, pharmaceuticals and toothpastes; also

as research tool in protein biochemistry.

Protein separation by SDS-PAGE can be used to estimate relative molecular mass, to

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

and the progress of a fractionation or purification procedure can be followed. Different

staining methods can be used to detect rare proteins and to learn something about their

biochemical properties. Specialized techniques such as Western blotting, two-

dimensional electrophoresis, and peptide mapping can be used to detect extremely

scarce gene products, to find similarities among them, and to detect and separate

isoenzymes of proteins (Caprette, 2012).

This experiment aims to subject a prepared bacterial protein isolate to SDS-PAGE.

Also, to separate protein molecules on the basis of molecular weight.

Results and discussion

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

with a positive charge will be attracted to the negatively charge node.

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

charged, regardless of the pH of the buffers.

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

about 10% of the glycine molecules are negatively charged.

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

shows the structure of SDS.

Figure 1 Structure of SDS

β−Mercaptoethanol, HO−(CH2)2 –Sh or in this experiment Dithiothreitol, is mixed in the

protein solution mainly to break the di-sulfide bonds in the protein which also help keep

the protein conformation. Figure 2 shows the reaction by β−Mercaptoethanol that

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

dissolved Oxygen removes the free radical.

Figure 3 decomposition of the persulfate ion

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

anode through the gel.

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

process. Therefore large volume of staining solution is recommended for use.

Figure photograph of the gel

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

slowly because of the loss of charge.

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

the neutral glycine.

Experimental methodology

A. PREPARATION OF BACTERIAL PROTEIN ISOLATE

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

in between washings. One hundred microliters of SDS-samples buffer on was

added and mixed and then was transferred to Eppendorf tube. The proteins were

denatured by boiling the tube for 5 minutes.

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.

Prepare separating gel solution: (Amount for 2 gels)

Water 4mL
1.5M Tris-HCl/0.4%, pH 8.8 3.3 mL
30% acrylamide/0.8% Bis 2.5 mL
10% SDS 100 µL
10% APS 60µL
5 TEMED 5.0 µl 4µL
 The solutions were added in a small beaker in the order given above.

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.

Prepare separating gel solution: (Amount for 2 gels)

Water 2.8 mL
0.5M Tris-HCl/0.4%, pH 6.8 1.26 mL
30% acrylamide/0.8% Bis pH 8.8 830 µL
10% SDS 50 µL
10% APS 50 µL
TEMED 5 µL

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.

C. LOADING THE SAMPLES AND RUNNING THE GEL

 While waiting for the stacking gel to polymerized, the sample and molecular

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.

Twenty microliters of each of the sample and 10 µL of the MW standards in a

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

corner of the gel.

D. Staining of SDS-PAGE Gels

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

gel was ready to photograph.

References

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