SDS-PAGE, Sodium Dodecyl Sulfate–PolyAcrylamide Gel

Electrophoresis–Animation
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SDS PAGE or sodium dot asyl sulfate polyacrylamide gel Electrophoresis is an electrophoresis method
and very powerful technique in the study of proteins.

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An SDS PAGE proteins are separated in a polyacrylamide gel based on their molecular weight.

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For sample preparation, a loading buffer containing SDS, beta, mercapto, ethanol, bromophenol,
BLUE, and glycerol is added to the protein samples.

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Protein samples may be biologically derived, for example, from prokaryotic or eukaryotic cells, tissues,
viruses, environmental samples, or purified proteins.

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When the sample buffer has been added, the samples are then heated at 95°C for 5 minutes.

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Proteins are large biomolecules consisting of one or more long chains of amino acid residues.

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Amino acids are organic compounds that contain a mined and carboxyl functional groups along with
a side chain.

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Proteins are formed by linking amino acids with peptide bonds and fold into one or more specific
spatial confirmations driven by a number of interactions such as hydrogen bonds, hydrophobic
interaction, desulfide bonds, and ionic bonds.

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SDS is an ionic surfactant and contain a polar head group with a net negative charge at the end of a
long hydrophobic carbon chain.

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SDS denatures the native proteins by disturbing the hydrogen bonds, hydrophobic and ionic
interactions, while the reducing agent Beta Mercoptothanol is used to cleave the disulfide bonds.

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Also, when a protein mixture is heated, SDS binds uniformly to the protein and the intrinsic charges of
this protein become negligible when compared to the negative charges contributed by SDS.
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This treatment brings the folded proteins down to linear molecules with net negative charge.

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Therefore, these proteins can be separated in a polyacrylamide gel based on their molecular weight.

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For gel preparation, A separating gel solution with pH 8.8 and a stacking gel solution with pH 6.8 are
used and both consist of acrylamide.

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This acrylamide, Ammonium persulfate and T Med.

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The gel is produced by polymerization between 2 glass plates anchored vertically in a cassette.

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The separating gel is poured first.

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The polymerization reaction is a vinyl addition polymerization initiated by a free radical generating
system.

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Ammonium persulfate is used as the free radical initiator, while T Med accelerates the rate of
formation of free radicals from persulfate.

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These free radicals convert acrylamide monomers to free radicals which react with unactivated
monomers to begin the polymerization chain reaction.

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The elongating polymer chains are randomly cross-linked by bisacrylamide, resulting in a gel with a
characteristic porosity.

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The pore size of the gel is related to the ratio of acrylamide to bisacrylamide.

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Low concentration is preferred for high molecular weight samples.

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The stacking gel solution is poured on top of the solid separating gel and a plastic comb placed on
the top of the stacking gel during polymerization enables the formation of small wells in the gel.

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Once the gel completely polymerizes, the comb is removed and the gel cassette is placed vertically
between 2 electrodes.

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Positive electrode located at the bottom of the gel whereas negative electrode positioned at the top
of the gel.

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The gel is inserted into a chamber and the Tris Glycine chloride buffer system with pH 8.3 is poured to
allow the conduction of current through the gel.

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SDS is also present in the gel and in the running buffer to make sure that once the proteins are
denatured, they stay that way throughout the run for sample application.

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In addition to the samples, a molecular weight size marker is usually loaded onto the gel.

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This consists of proteins of known sizes and thereby allows the estimation of the sizes of the proteins
in the actual samples, which migrate in parallel in different tracks of the gel.

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Each sample is pivoted into its own well in the gel, and as we have seen previously, bromophenol blue
and glycerol are present in the samples.

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Bromophenol Blue is a dye that is useful for visualizing the sample in the well, and glycerol increases
the density of a sample and used to make the sample fall to the bottom of the sample well rather than
just flow out and mix with the running buffer.

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After the sample application procedure, an electric field is applied across the gel, causing the
negatively charged proteins to migrate across the gel away from the negative electrode and towards
the positive electrode.

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Also, small proteins migrate relatively easily through the mesh of the gel, while larger proteins are
more likely to be retained and thereby migrate more slowly through the gel.

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The stacking gel is used to ensure that all of the proteins arrive at the separating gel at the same time.

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Because gel wells are around 1cm deep, so in the absence of a stacking gel, proteins would all enter
the separating gel at different times resulting in very smeared bands.
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As we have seen previously, the running buffer consists of glycine and chloride ions, so in the sample
wells we will have proteins, chloride ions and glycine.

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Proteins and chloride ions are negatively charged.

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Well glycine can exist in three different charge states depending on the pH.

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The isoelectric pointer Phi of glycine is equal 5.97.

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At this pH, glycine carries neutral charge do the gain and loss of protons.

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At aph below its Phi, glycine carries a net positive charge and at Aph above its Phi it becomes
negatively charged.

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The running buffer pH is equal 8.3 which above the Phi of glycine.

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So at this pH glycine is negatively charged.

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Once the electric current is applied, proteins, chloride ions and glycine are forced to enter the stacking
gel, where the pH is equal 6.8.

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At this pH, glycine switches predominantly to the zwitterionic neutrally charged state.

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This loss of charge causes them to move very slowly in the electric field.

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The chloride ions, on the other hand, move much more quickly.

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Chloride ions migrate in front of the proteins as leading ions, and glycine molecules migrate behind
the proteins as initial trailing ions.

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Consequently, proteins are trapped and concentrated into a narrow zone between the chloride ions
and glycine.
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This procession carries on until it hits the separating gel, where the pH switches to 8.8.

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At this pH, the glycine molecules are mostly negatively charged and can migrate much faster than the
proteins.

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Proteins are separated, so higher molecular weight proteins move more slowly through the porous
acrylamide gel than lower molecular weight proteins.

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Due to the relatively small molecule size of bromophenol blue, it migrates faster than proteins and by
optical control of the migrating colored band, the electrophoresis can be stopped before the dye and
before the samples have completely migrated through the gel and leave it at the end of the
electrophoretic separation.

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All proteins are sorted by size and can then be analyzed by other methods, for example protein
staining methods and immunological detection such as the Western Blot technique.

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After removing the glass plates, the stacking gel is discarded and the separating gel can be stained.

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Camasi blue is the most popular protein stain.

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It is an inonic dye which non specifically binds to proteins.

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The structure of Camasi blue is predominantly non polar and it is usually used in methanolic solution
acidified with acetic acid.

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Proteins in the gel are fixed by acetic acid and simultaneously stained.

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After staining, the proteins are detected as blue bands on a clear background.

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The molecular weight size markers in a separate lane in the gel can be used to determine the
approximate molecular mass of unknown biomolecules by comparing the distance traveled relative to
the marker.