Task Two Electrophoresis .pdf
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Title: Task Two - Electrophoresis
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Task Two - Electrophoresis
In the early 1900s, a Russian botanist named Mikhail Tswett became interested in the
individual chemical compounds in plants. He noted that extracting ground-up plant
material extracts with different solvents produced different coloured solutions. One of his
experiments involved pouring a plant extract through a glass tube packed with powdered
calcium carbonate. As the liquid passed through the solid powder, bands of colour
appeared; these were the individual compounds, separated from each other by the
interaction of the solid (which remained fixed in the tube) and the liquid extract (which
flowed through the tube and out the other end). Tswett had invented chromatography, a
word he derived from the Greek words for colour (chroma) and writing (graphe).
Chromatography is a method of separating a mixture into its component parts, based on
how large/small the parts are. Coloured texta can be split into its separate colours of
which it is made up. This works because the dye is taken up with the water through the
fibres of the paper and with this information we can prove that the smaller molecules of
dye will travel further meanwhile the larger molecules do not travel as far. This is the
same with DNA the smaller chains of nucleotide bases will end up further along the
agarose gel when the larger chains of nucleotide bases will not get as far.
For gel electrophoresis you need agarose gel. An agarose is a polysaccharide polymer
material, generally extracted from seaweed. Agarose is available as a white powder
which dissolves in near-boiling water, and forms a gel when it cools. Standard agaroses
derived from Gelidium has a gelling temperature of 34–38 °C and a melting temperature
of 90–95 °C, while those derived from Gracilaria, due to its higher methoxy substituents,
has a gelling temperature of 40–52 °C and melting temperature of 85–90 °C. Agarose is
a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide
made up of D-galactose and 3,6-anhydro-L-galactopyranose.
Cohesion - or cohesive attraction or cohesive force is the action or property of like
molecules sticking together, being mutually attractive. Like mercury in a glass container
the meniscus faces downwards.
Adhesion - is the tendency of dissimilar particles or surfaces to cling to one another
(cohesion refers to the tendency of similar or identical particles/surfaces to cling to one
another). Like water in a glass container the meniscus faces upwards.
Electrophoresis occurs because particles dispersed in a fluid almost always carry an
electric surface charge. An electric field exerts electrostatic Coulomb force on the
particles through these charges. Recent molecular dynamics simulations, though,
suggest that surface charge is not always necessary for electrophoresis and that even
neutral particles can show electrophoresis due to the specific molecular structure of
water at the interface.
Agarose gel electrophoresis is a method used in biochemistry and molecular biology to
separate DNA, or RNA molecules by size. This is achieved by moving negatively
charged nucleic acid molecules through an agarose matrix with an electric field
(electrophoresis). Shorter molecules move faster and migrate farther than longer ones.
Gel electrophoresis is used in many different situations like DNA profiling, therapy,
genetic testing, agriculture, cloning, conservation.
DNA profiling is a technique by which individuals are identified on the basis of their
respective DNA profiles. Within the non-coding region of an individual's genome, there
exists satellite DNA - long stretches of DNA made up of repeating elements called short
tandem repeats (STRs). These repeating sequences can be excised to form fragments,
by cutting with a variety of restriction endonuclease (which cut DNA at specific sites). As
individuals all have a different number of repeats in a given sequence of satellite DNA,
they will all generate unique fragment profiles. These different profiles can be compared
using gel electrophoresis.
Another use for electrophoresis is electrotherapy. It allows shallow to deliver into the
layers of skin - the dermis and epidermis medicine, where the medicine is absorbed
through tiny vessels in the lymph and blood. Once in the lymph current and flow, it is
delivered to all tissues and organs, but the greatest concentration remains on the area
where the medicine is injected. The effect of the medicine and the electric current can
reduce the dose of the drug, because even at low concentrations of the substance is a
therapeutic effect. With the help of electric current increases the activity of the medicine,
which promotes the use of low dosages.
Gel electrophoresis can also be used to find genes related to a disease, for example
premature menopause. If the fragments are found only in people who have the disease,
it suggests that the fragments contain the DNA from a gene variant that might mean a
person is more susceptible to getting the disease. The picture shows the results of DNA
analysis from seven different people. The first lane (1) represents a ‘ladder’, which
allows you to determine the size of the DNA fragments that have been separated.
Imagine that the DNA loaded in wells 2, 3, 4 and 5 comes from patients with the disease,
and DNA loaded in wells 6, 7, 8 and 9 comes from people who do not have the disease.
Three of the four patients with the disease have an extra band. The person with DNA
loaded in lane 2 also has the disease, but the results do not show the same banding
pattern as other people with the disease. This suggests that more than one genetic
variation may be associated with this disease.
Vaccine analysis is another important application of electrophoresis. There are several
vaccines that have been purified, processed and analysed through electrophoresis, such
as the influenza vaccine, hepatitis vaccine and polio vaccine. The exact steps done in
the vaccine analysis, however, cannot be determined due to confidentiality reasons of
the pharmaceutical companies. Nevertheless, data reports from vaccine manufacturers
such as Wyeth, Merck and Sanofi-Aventis presents electrophoresis as an effective
vaccine analysis method.
^ Just after the dyes were inserted into the wells. ^
^ Just as the power pack was attached to the electrophoresis tank. ^
^ Bubbles from the negative end of the electrophoresis tank. ^
^ The dyes moving and splitting up into each colour it is made up of (15-20 minutes into
the power being on. ^
^ Second progress pic, almost ready for removal and measuring. ^
^ The dyes split well enough for measuring after around 30-40 minutes (blurry because
the camera was focusing on the reflection as the dyes always look out of focus . ^
Well Number (Left to
Right + Wells being at
(dye colour code)
Dyes Used Within
Number of Colours
Split into Upon
1 - Yellow
1 - Orange
1 - Blue
N/A - Too clear to see
1 - Pink
L - Ladder
Equal parts of each dye
B, OG, RB, T, and IC
Test Combinations for
Tartrazine/ Orange G
2 - Yellow + Orange
Indigo Carmine/ Rose
1 - Pink only as Indigo
Carmine was too clear
Colours Measured at the End
Distance Travelled (Largest to Smallest +
Measured with Ruler on the Gel)
With all of the data we can see how yellow has the largest migration, while pink had the
shortest, the two test combinations used easily and clearly split between the colours
aside from 4, with 4 you could not see the Indigo Carmine as it was once again too clear
to be seen much like its counterpart IC. For 3 the split was easily distinguishable as the
shades between the yellow and orange were enough to differentiate between the two
colours. The ladder wasn’t exactly too successful as it lacked a clearly visible colour
- the Rose Bengal (pink) - although the IC dye may be present but as it is transparent we
cannot see. We could deduce the dyes included in both the 3rd and 4th test sample had
Tartrazine/ Orange G and Indigo Carmine/ Rose Bengal respectively because if you
compare those wells to the previous know dyes you can see for example that the colours
in well 1 and 2 were visible in sample 3. We can do the same for sample 4 but without
the inclusion of Indigo Carmine, as there was nothing to be compared.
We can also deduce from the results that yellow dye was the smallest while as it
travelled the furthest (2.5cm), orange close second with a distance of 2cm, blue coming
4th with a distance of 1.4cm, and finally pink being the largest molecule with a distance
of 1cm. This is because the fibres of the paper restrict the speed of which the larger
molecules can travel, like trying to fit something very large down a small hallway while
the smaller molecules travel through with ease.
There are three kinds of gel agarose, polyacrylamide, and starch.
Agarose gels are made from the natural polysaccharide polymers extracted from
seaweed. Agarose gels are easily cast and handled compared to other matrices,
because the gel setting is a physical rather than chemical change. Samples are also
easily recovered. After the experiment is finished, the resulting gel can be stored in a
plastic bag in a refrigerator. Agarose gels do not have a uniform pore size, but are
optimal for electrophoresis of proteins that are larger than 200 kDa. Agarose gel
electrophoresis can also be used for the separation of DNA fragments ranging from 50
base pair to several megabases (millions of bases), the largest of which require
specialized apparatus. The distance between DNA bands of different lengths is
influenced by the percent agarose in the gel, with higher percentages requiring longer
run times, sometimes days. Instead high percentage agarose gels should be run with a
pulsed field electrophoresis (PFE), or field inversion electrophoresis.
Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins ranging in
size from 5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide
gel. Pore size is controlled by modulating the concentrations of acrylamide and bisacrylamide powder used in creating a gel. Care must be used when creating this type of
gel, as acrylamide is a potent neurotoxin in its liquid and powdered forms. Traditional
DNA sequencing techniques such as Maxam-Gilbert or Sanger methods used
polyacrylamide gels to separate DNA fragments differing by a single base-pair in length
so the sequence could be read. Most modern DNA separation methods now use
agarose gels, except for particularly small DNA fragments. It is currently most often used
in the field of immunology and protein analysis, often used to separate different proteins
or isoforms of the same protein into separate bands. These can be transferred onto a
nitrocellulose or PVDF membrane to be probed with antibodies and corresponding
markers, such as in a western blot.
Partially hydrolysed potato starch makes for another non-toxic medium for protein
electrophoresis. The gels are slightly more opaque than acrylamide or agarose. Nondenatured proteins can be separated according to charge and size. They are visualised
using Napthal Black or Amido Black staining. Typical starch gel concentrations are 5% to
Another factor for how the migration is affected is protein shape. Non-reduced protein
will completely unfold, retaining somewhat globular shape. It will also bind less SDS
compared to its mass - normally it will run faster, but not always. Heavily glycosylated
protein will remain somewhat globular, even when reduced, as the glyco branches
cannot be linearised - and should migrate faster then a protein with the same mass
made only from amino acids.
The results in the experiment were not really expected on the basis of not knowing how
electrophoresis works before this experiment. I expected the dyes to split into their
colours as that is what gel electrophoresis is for but the extent of which they split and
what colours travel what distance was unknown. Linear DNA can be resolved by size
using agarose gels of various concentrations. The greater the percentage of agarose,
the smaller the linear DNA that can be resolved. The sugar polymers that make up the
agarose gel matrix (powdered agarose heated in appropriate buffer, poured into a gel
tray and allowed to solidify) act like a sieve. The greater the agarose concentration, the
smaller the pores created in the gel matrix, and the more difficult it is for large linear DNA
molecules to move through the matrix. Changing the agarose concentration changes the
size of the sieve matrix of the gel. However, there is an upper and lower limit to accurate
separation of DNA molecules using agarose gel electrophoresis. The experiment ran
very smoothly as I was the only person conducting it and it involved no participants. The
experiment was highly successful as the procedure went very well. There were no
problems with any equipment or any extraneous variables which could have affected the
experiment. The weather was fine as the experiment was conducted inside.
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