ELECTROPHORESIS
Definition
"Electrophoresis" refers to the electromotive force (EMF) that is used to move the molecules
through the gel matrix. By placing the molecules in wells in the gel and applying an electric
field, the molecules will move through the matrix at different rates determined largely by their
mass when the charge to mass ratio (Z) of all species is uniform, toward the anode if negatively
charged or toward the cathode if positively charged.
It is one of the most popular ways to analyze macromolecules such as proteins, DNA and RNA.
There are several types of electrophoresis, but the concepts are similar. The machine has an
anode (positive charge) and a cathode (negative charge). Negative ions move toward the anode,
and positive-charged ions move towards the cathode. The rate and distance traveled by these
molecules help scientists classify and study different biomolecules.
Several alternative types of gel electrophoresis exist and can be classed as one-dimensional such
as agarose gels, polyacrylamide gel electrophoresis (native or SDS-PAGE) and
Isoelectricfocusing (IEF) or two dimensional such as 2D-PAGE. Gel electrophoresis can also be
carried out using narrow gauge capillaries.
Types
1. Agarose Gels
Agarose gels are an electrophoresis method to separate RNA and DNA molecules. It separates
the molecules based on charge and size. DNA molecules are negatively charged, so they move
through the gel quickly depending on size. Smaller DNA fragments move more quickly than
larger ones due to friction resistance.
2. PAGE (Polyacrylamide Gel Electrophoresis)
a) SDS-PAGE
SDS-PAGE (Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis) is a common form
of electrophoresis for analyzing proteins. The SDS part of the name is a protein denaturing
detergent that causes the molecule to unfold. The detergent binds to the polypeptide in a 1:1 ratio
with each segment of the protein to give it a charge. The protein polypeptides move through the
gel at different rates depending on mass, allowing researchers to study proteins based on size.
b) Native-PAGE
Native gels are similar to SDS-PAGE, except the detergent (SDS) is not used to denature
proteins. Native gels are only able to separate proteins up to 2,000 kDa in size. Because the
proteins are left folded, the dyes used are also different than SDS-PAGE.
3. Electrofocusing
Electrofocusing takes advantage of charge and pH values of proteins. A container is filled
with a gel solution that has an increasing pH gradient. The amino acids that form polypeptides
have different acidic or basic charges. The protein travels through the gel, obtaining or losing
protons depending on its charge. As the protein particle moves through the gel, it eventually
becomes neutral and gets stuck in an isoelectric position.
4. 2-DIMENSIONAL GEL ELECTROPHORESIS
Using this technique, proteins are separated by two different properties. Initially proteins
or polypeptides are separated on the basis of their net charges by isoelectric focusing. Gels are
then turned 90° and separation continues based on their molecular weight. Because it is unlikely
that two molecules will be similar in both properties, molecules are more effectively separated in
2-D electrophoresis than in 1-D electrophoresis gels.
In 2-D electrophoresis, proteins are initially separated by isoelectric focusing. This
isoelectric focusing occurs across a pH gradient. The protein band will stop moving across the
gel at its isoelectric point when the charge associated with the different amino groups is nullified
by the pH. The gel is then turned 90o and the second electrophoresis occurs using SDS to
separate the proteins by molecular weight. This second dimension of focusing gives a series of
spots across the gel and each spot is a specific protein.
Typical representation of a 2-D SDS-PAGE with proteins separated by charge and
molecular weight.
5. Capillary
Capillary electrophoresis is a method similar to SDS-PAGE. It separates molecules based on
their charge and mass. Molecules are placed in rows called capillaries filled with conductive,
electrolyte fluid. The analytes move in a speed relative to their charge and mass. This method is
an older technique introduced in the 1960s that’s why SDS-PAGE is usually preferred in labs.
Electroendosmosis
What is Electroendosmosis (EEO)?
A requirement of the gel is that it is neutral and that included the absence of charged impurities.
If the gel contains any charged groups then an effect known as electroendosmosis will take place.
As these charged groups are immobilised onto the gel they cause a solvent flow towards one of
the electrodes, usually the cathode (negative) and thus in opposition to the sample flow. This is
obviously undesirable as this will slow down and may distort the migration of the samples
reducing resolution.
Principle of Electroendosmosis
The static support, the stabilizing medium (e.g. the gel) and/or the surface of the separation
equipment such as glass plates, tubes or capillaries can carry charged groups: e.g. carboxylic
groups in starch and agarose, sulfonic groups in agarose, silicium oxide on glass surfaces. These
groups become ionized in basic and neutral buffers: in the electric field they will be attracted by
the anode. As they are fixed in the matrix, they can not migrate. This results in a compensation
by the counterflow of H3O+ ions towards the cathode: electroendosmosis.
In gels, this effect is observed as a water flow towards the cathode, which carries the solubilized
substances along. The electrophoretic and electroosmotic migrations are then additive. The
results are: blurred zones, and drying of the gel in the anodal area of flatbed gels. When fixed
groups are positively charged, the electroosmotic flow is directed towards the anode.
Applications
Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and
biochemistry. The results can be analyzed quantitatively by visualizing the gel with I-IV light
and a gel imaging device. The image is recorded with a computer operated camera, and the
intensity of the band or spot of interest is measured and compared against standard or markers
loaded on the same gel. The measurement and analysis are mostly done with specialized
software. Depending on the type of analysis being performed, other techniques are often
implemented in conjunction with the results of gel electrophoresis, providing a wide range of
field-specific applications.
DNA CHECK BY AGAROSE GEL ELECTROPHORESIS
PRINCIPLE:
Agarose gel electrophoresis separates DNA fragments according to their size. An electric current
is used to move the DNA molecules across an agarose gel, which is a polysaccharide matrix that
functions as a sieve to help "catch" the molecules as they are transported by the electric current.
The phosphate molecules that make up the backbone of DNA molecules have a high negative
charge. When DNA is placed on a field with an electric current, these negatively charged DNA
molecules migrate toward the positive end of the field, which in this case is an agarose gel
immersed in a buffer bath. The agarose gel is a cross-linked matrix i.e., a three-dimensional
mesh or screen. The DNA molecules are pulled to the positive end by the current, but they
encounter resistance from this agarose mesh. The smaller molecules are able to navigate the
mesh faster than the larger ones. This is how agarose electrophoresis separates different DNA
molecules according to their size. The gel is stained with ethidium bromide so as to visualize
these DNA molecules resolved into bands along the gel. Ethidium bromide is an intercalating
dye, which intercalate between the bases that are stacked in the center of the DNA helix. One
ethidium bromide molecule binds to one base. As each dye molecule binds to the bases the helix
is unwound to accommodate the stain from the dye. Closed circular DNA is constrained and
cannot withstand as much twisting strain as can linear DNA, so circular DNA cannot bind as
much dye as can linear DNA. Unknown DNA samples are typically run on the same gel with a
"ladder." A ladder is a sample of DNA where the sizes of the bands are known. Unknown
fragments are compared with the ladder fragments (size known) to determine the approximate
size of the unknown DNA bands. Approximately 10ng is visible in a single band on a horizontal
agarose gel.
MATERIALS:
•Agarose
•TAE buffer
•Gel casting tray, comb, power pack
•Sample DNA
•Loading dye
•Sterile micro tips
•EtBr staining solution
•UV transilluminator or Gel Documentation System
INSTRUCTIONS:
For casting gel, agarose powder is mixed with electrophoresis buffer (TAE) to the desired
concentration, and then heated in a microwave oven until completely melted. After cooling the
solution to about 60°C, it is poured into a casting tray containing a comb and allowed to solidify
at room temperature for nearly 45 min.
After the gel has solidified, the comb is removed, using care not to rip the bottom of the wells.
The gel, still in its plastic tray, is inserted horizontally into the electrophoresis chamber and just
immersed with buffer (TAE). DNA samples mixed with loading buffer are then pipeted into the
sample wells, the lid and power leads are placed on the apparatus, and a current is applied. The
current flow is confirmed by observing bubbles coming off the electrodes. DNA will migrate
towards the positive electrode, which is usually colored red. The distance DNA has migrated in
the gel can be judged by visually monitoring migration of the tracking dyes. Bromophenol blue
and xylene cyanol dyes migrate through agarose gels at roughly the same rate as double-stranded
DNA fragments of 300 and 4000 bp, respectively. When adequate migration (2/3 of the gel) has
occurred, DNA fragments are visualized by staining with ethidium bromide. This fluorescent dye
intercalates between bases of DNA and RNA. It is often incorporated into the gel so that staining
occurs during electrophoresis, but the gel can also be stained after electrophoresis by soaking in a
dilute solution of ethidium bromide. To visualize DNA or RNA, the gel is placed on a ultraviolet
transilluminator. Be aware that DNA will diffuse within the gel over time, and examination or
photography should take place shortly after cessation of electrophoresis.
Preparation of 1% Agarose gel:
Weigh 0.50 g agarose; add in 50 ml 1X TAE and melt agarose in a microwave oven for 2-3 min.
Cool down to about 45 to 50°C (bearable warmth) and pour into the gel platform with the comb
in position.
Running gel:
After solidification of the gel (approx. 45 min), place the gel in a gel tank with 1 X TAE buffer.
Buffer should be filled to the surface of the gel. Load the samples in the well and run the gel at
70 V till the blue dye runs to the end.
Staining the gel:
Prepare staining solution by adding 10 µl of 10 mg/ml stock of Ethidium bromide in 100 ml of
Distilled water. Place the gel in staining solution for 30 min and view the gel in UV
transilluminator.
Fig 1: Agarose Gel Electrophoresis Method
10X TAE (pH 8.5): 1000 ml
Tris-Base– 48.4 g
EDTA – 7.44 g
Glacial Acetic Acid – 11.42 mL
Dissolve in 600 ml of dd water. First allow the Tris to dissolve in water, and then add EDTA.
Make up the volume to one liter and autoclave. (Check and confirm the pH is about 8.5).
Ethidium Bromide Stock:
Stock 10 mg/ml. working concentration 1 µg/ml.
a. Agarose Concentration: Agarose is a linear polymer that is derived from red algae. By using
gels with different concentrations of agarose, one can resolve different sizes of DNA fragments.
Higher concentrations of agarose facilitate separation of small DNAs, while low agarose
concentrations allow resolution of larger DNAs. The image in the right shows migration of a set
of DNA fragments in three concentrations of agarose, all of which were in the same gel tray and
electrophoresed at the same voltage and for identical times. Notice how the larger fragments are
much better resolved in the 0.7% gel, while the small fragments separated best in 1.5% agarose.
The 1000 bp fragment is indicated in each lane. Concentration of agarose and its relation with
DNA size can easily be understood from the following table.
b. Voltage: As the voltage applied to a gel is increased, larger fragments migrate proportionally
faster that small fragment. For that reason, the best resolution of fragments larger than about 2 kb
is attained by applying no more than 5 volts per cm to the gel (the cm value is the distance
between the two electrodes, not the length of the gel).
b. Electrophoresis Buffer: Several different buffers have been recommended for
electrophoresis of DNA. The most commonly used for duplex DNA are TrisborateEDTA (TBE = 90 mM Tris HCI, 20 mM boric acid, 2 mM EDTA, pH 8.3) and Trisacetate (TAE = 40 mM Tris HCI, 20 mM Na acetate, 1.8 mM EDTA, pH 7.8). DNA
fragments will migrate at somewhat different rates in these two buffers due to differences
in ionic strength. TAE is used for preparative separations when the gel is to be dissolved
with NaCl solutions prior to binding of the DNA with glass powder. TBE is used for
most analytical work. However for separation of large DNA fragments, electrophoresis at
a low voltage gradient in TAE buffer is optimal, due to the low extent of diffusion of
large molecules .Buffers not only establish a pH, but provide ions to support
conductivity. If you mistakenly use water instead of buffer, there will be essentially no
migration of DNA in the gel! Conversely, if you use concentrated buffer (e.g. a 10X
stock solution), enough heat may be generated in the gel to melt it.
c. Difference b/w TAE & TBE buffer
i. Recovery of nucleic acid
TAE has been prefavorably used so far because of the poor recovery of nucleic acids
from TBE-gel. However, recent commercial recovery kits utilizing silica works well for
either TAE-gel or TBE-gel.
ii.Buffering Capacity: TBE has more buffering capacity than TAE
iii. Moving Rate: Since ion strength of TBE is higher than TAE, solutes in TBE move
faster than in TAE
d. Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates
between bases of nucleic acids and allows very convenient detection of DNA fragments in gels,
as shown by all the images on this page. As described above, it can be incorporated into agarose
gels, or added to samples of DNA before loading to enable visualization of the fragments within
the gel. As might be expected, binding of ethidium bromide to DNA alters its mass and rigidity,
and therefore its mobility.