overview of electrophoresis
TRANSCRIPT
OVERVIEW OFELECTROPHORESIS
V. MAGENDIRA MANI
ASSISTANT PROFESSOR
PG & RESEARCH DEPARTMENT OF BIOCHEMISTRY
ISLAMIAH COLLEGE (AUTONOMOUS)
VANIYAMBADI
https://tvuni.academia.edu/mvinayagam
Electrophoresis
Electrophoresis is defined as the migration of charged particles
through a solution under influence of an electric field.
Biological molecules such as amino acids, peptides, protein,
nucleotides, and nucleic acids possess ionisable groups and are
made to exist as electrically charged species either as cations or
anions. Even carbohydrates can be given weak charges by
derivatization such as borates or phosphates. In electrophoresis
cation move towards cathode and anion move towards anode.
The rate of migration is depends on
The charge of the particle Applied electric field
Temperature
Nature of the suspended medium
FACTORS AFFECTING ELECTROPHORESIS
SAMPLE
Charge : Rate of migration increases with increase in
net charge. It depends on pH.
Size : Rate of migration decreases for larger
molecules. It is due to increase frictional and electrostatics
forces.
Shape : Molecular have similar charge but differ in
shape exhibit different migration rate.
ELECTRIC FIELD
According to ohms law
I=V/R
Current = Voltage / Resistance
Voltage : Increase in voltage leads to increase in rate of
migration
Current : Increase in current leads to Increase in
voltage, so the migration also Increases
Resistance: If resistance increase migration decreases.
BUFFER
Buffer determines & stabilizes pH of the supporting medium
Also affects the migration rate of compounds in a number of ways
Composition of Buffer
Acetate
Barbiturate Citrate
EDTA Formate
Phosphate
Pyridine buffers commonly used.
IONIC STRENGTH
As ionic strength of buffer increases
Proportion of current carried by buffer increases
Proportion of current carried by the sample decreases and
hence showing decrease in sample rate of migration.
High ionic strength
• Also increases overall current and hence heat is produced
As ionic strength of buffer decreases
Proportion of current carried by buffer decreases
Proportion of current carried by the sample increases and
hence showing increase in sample rate of migration.
Low ionic strength
• Also decrease in overall current and hence decrease in heat
production
pH
pH determines the ionization, if ionization of organic acid
increases as pH increases, ionization of organic acid
decreases as pH decrease.
Therefore the degree of ionization is pH dependent.
SUPPORTING MEDIUM
Adsorption
Adsorption is the retentio n of sample molecule by
supporting medium.
Adsorption causes tailing of sample so that it moves in the
shape of a ‘comet’ rather than a distinct compact band
Adsorption reduces both the rate of migration and resolution
of separation of molecule.
Electro – endo-osmosis
Electro – endo-osmosis due to the presence of charged groups on
the surface of the supporting medium
Eg. Paper - Carboxyl group (COO-)
Agarose - Sulphate group (SO2-)
Glass wall- Silanol (SiO-)
.
Above the pH value of three these charged groups will
have ionize and generates negatively charged sites. These
ionized groups create an electrical double layer or region at
supporting medium.
When voltage is applied, cation in electrolyte near
supporting medium migrate towards cathode pulling
electrolyte solution with them. This creates a net Electro –
endo-osmotic flow towards the cathode.
The Electro – endo-osmosis will accelerate the movement
of cations, but retard anion movements.
MOLECULAR SIEVING
Gels have sieve like structure
In agar, starch, and poly acryl amide gels the movement of
large molecule is hindered by decreasing the pore size,
since all the molecule has to transverse through pores.
If sephadex gel is used, small molecules are tightly held
by pores and large molecules are excluded by small pores
causing movement outside the pores called molecular
sieving.
TISELIUS MOVING
BOUNDARY
ELECTROPHORESIS
Moving boundary electrophoresis
Moving boundary electrophoresis technique was introduced by
Swedish biochemist Arne Tiselius (1937) to separate proteins. The
Tiselius apparatus is an U–tube in which the protein dissolved in
buffer is taken in the lower part of the tube and plain buffer taken
in the upper part of the tube. Then the limbs are connected to
electrodes. Electrophoresis is carried out for 21/2 hours.at a
reduced temperature (40 C). As current passed protein moved
towards limbswhere only buffer is placed. Depending on the
charge, the rate of movement differs and they will form
boundaries. The movement of boundaries is observed in schieleran
optical system.
As protein moves, the system gives peaks. Each peak indicates
one protein. Optical system works in the variation of refractive
index of each boundary with the help of this diagram one can find
the concentration & direction of various peaks.
Applications:-
To separate proteins.
To study protein–protein interaction.
To measure homogenesity of protein. Homogenous protein
give one peak.
Disadvantages:-
Slow technique.
Requires complex optical system.
Liable to disturbances by conventional and vibrational current.
Cannot give complete separation of one protein.
SODIUM DODECYL
SULPHATE POLY ACRYL
AMIDE GEL
ELECTROPHORESIS
Principle
SDS PAGE is widely used method for separating
protein mixture and for determining the molecular weights.
SDS - is an anionic detergent
Binds strongly to protein
Causes their Denaturation
Polymerization
Cross linked poly acryl amide gel are formed by
polymerization of acryl amide monomer and N, N’ Methylene
Bisacryl amide in the presence of ammonium per sulphate and
TEMED (Tetra Methylene Diamine). Ammonium per sulphate
acts as free radical catalyst and TEMED acts as initiator.
Photopolymerization
Photopolymerization is an alternative method that can be
used to polymerise Acryl amide gels. Ammonium per
sulphate, TEMED are replaced by Riboflavin. When the
gel is poured it is placed in front of bright light for 2 – 3
hrs. Photopolymerization of riboflavin generates a free
radical that initiates polymerization.
Pore size of the PAGE
Pore size of the gel can be varied by changing the
concentration of both Acryl amide and Methylene Bisacryl
amide. PAGE can be made with content of 3 % to 30 %. Low
percentage of gel 3 % have large pore size and used for
separation of proteins and DNA. High percentage of gel 10 -20
% have smaller pore size and used for SDS PAGE.
Sample preparation
The sample is boiled with the buffer containing β-
mercaptoethanol and SDS for 5 min. β-mercaptoethanol
reduces any disulfide bridges in protein that are holding
tertiary structure of protein.
SDS binds strongly to proteins and denature the protein
On average one SDS molecule binds to every two amino
acids. So the native charge of the molecule is completely
swamped by SDS molecule (negative)
Each protein molecule will be fully denatured, opens in to
a rod shaped structure with a series of – ve charged SDS
molecule along with polypeptide chain.
Gel preparation Gels used are vertical slabs, because it is more economical and
more sample can be compared with each other when run under
identical conditions (eg. 20 different samples)
Gels are prepared in glass containers in which they are to be
used. The two glass plates are held together but held apart from
each other by plastic spacer, vertical slab gels are run along with
the glass plate.
Choice of percentage of gel to be used depends on the size of
the protein sample. Separating gel used may vary from 10 %
PAGE to 15 % PAGE. 15 % of gel used for separation of protein
having molecular weight 10,000 to 1, 00,000 and 10 % of gel
used for separation of protein having molecular weight 1,
50,000.
Sample application
Dissolved samples can be applied using a micro syringe into wells
of the gel.
Sample buffer containing 10-15 % Sucrose or Glycerol, which
increase the density of the buffer and ensures the sinking of the
sample in to the wells.
It also prevents the sample from mixing with buffer in the upper
buffer reservoir.
Sample buffer contain marker/tracker dye Bromophenol blue. It
is a small molecule and it moves freely and indicates the
electrophoretic migration.
Urea, SDS, Disulfide reducing agent such as β-Mercaptoethanol
are added to protein sample to facilitate their solubilisation.
Protein sample can be loaded in the form of sharp band by using
a staking gel over the separating gel.
Only µ g of sample are used for analyzing.
Running the gel
The gel slab sandwiched in between the glass plate is placed in
the lower reservoir with the top of the gel in contact with the
buffer in the upper reservoir.
Thus the gel completes the electrical circuit between the lower
and upper compartments. Although the buffer dissipates the heat
generated, additional cooling may be needed.
In sample small protein can more easily pass through the pores
and larger proteins are successively retarded by frictional
resistance due to sieving effect of the gel.
Precise voltage and time required for the optimal separation
Voltage: 30 mA; Time; 3 hrs
Detection
When tracker dye reaches the bottom of the gel the current is
turned off.
Gel slabs are removed without any pressure, after removal
gel is immersed in 7 % acetic acid to minimize diffusion of
components.
Then the gel is shaken well in an appropriate stain solution.
Usually Commassive Brilliant Blue R250 is used and the gel
is immersed for few hours.
Then the gel is transferred in to a destain solution and kept
for overnight.
Destain solution removes unbound background dye from gel
leaving stain protein visible as blue bands on a clear
background.
Staining solution (100 ml)
a. Coomassie Brilliant Blue R-250: 250 mg
b. Methanol: 50 %
c. Acetic acid: 10 %
d. Distilled water: 40 %
De-staining solution (100 ml)
a. Methanol: 50 %
b. Acetic acid: 10 %
c. Distilled water: 40 %
Time required for electrophoresis
Gel preparation - 1– 1 ½ hrs
Running the gel - 3 hrs
Staining - 2– 3 hrs
De-staining - overnight
Determination of Molecular weight (PROTEIN) by SDS
PAGE.
The Molecular weight can be determined comparing mobility
of standard protein of known Molecular weight with of
unknown Molecular weight that is run on the same gel.
A calibration curve is constructed for standard protein of
known Molecular weight by
Distance migrated Vs Molecular weight x 104
The migration of unknown is measured are extrapolating this
value in the calibration curve, the molecular weight of
unknown can be determined.
AGAROSE GEL
ELECTROPHORESIS
Agarose gel electrophoresis is the easiest and most popular way of
separating and analyzing DNA. Here DNA molecules are
separated on the basis of charge by applying an electric field to
the electrophoretic apparatus. Shorter molecules migrate more
easily and move faster than longermolecules through the pores of
the gel and this process is called sieving. The gel might be used to
look at the DNA in order to quantify it or to isolate a particular
band. The DNA can be visualized in the gel by the addition of
ethidium bromide.
Agarose is a polysaccharide obtained from the red algae
Porphyra umbilicalis. Its systematic name is (1 4)-3, 6-
anhydro-a-L-galactopyranosyl-(1,3)-β-D-galactopyranan.
Agarose makes an inert matrix. Most agarose gels are
made between 0.7% and 2% of agarose. A 0.7% gel will
show good separation for large DNA fragments (5-10kb)
and a 2% gel will show good resolution for small
fragments with size range of 0.2-1kb.
Materials Required:
Gel casting trays, which are available in a variety of sizes and
composed of Uv transparent plastic.
Sample combs, around which molten agarose is poured to
form sample wells in the gel.
Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or
Tris-borate-EDTA (TBE).
The migration rate of DNA fragments in both of these buffers is
somewhat different due to the differences in ionic strength. These
buffers provide the ions for supporting conductivity.
Loading buffer, which contains something dense (e.g. glycerol)
to allow the sample to "fall" into the sample wells, and one or two
tracking dyes, which migrate in the gel and allow visual
monitoring or how far the electrophoresis has proceeded.
Ethidium Bromide (EtBr)
Ethidium bromide, a fluorescent dye used for staining nucleic
acids. It is an intercalating agent which intercalates between
nucleic acid bases and allows the convenient detection of DNA
fragments in gel. When exposed to UV light, it will fluoresce
with an orange colour. After the running of DNA through an
EtBr-treated gel, any band containing more than ~20 ng DNA
becomes distinctly visible under UV light. EtBr is a known
"mutagen", however, safer alternatives are available. It can be
incorporated with agarose gels or DNA samples before loading,
for visualization of the fragments. Binding of Ethidium bromide
to DNA alters its mass and rigidity, and thereby its mobility.
Transilluminator (an ultraviolet light box), which is used to
visualize ethidium bromide stained DNA in gels.
General procedure
Casting of gel
The gel is prepared by dissolving the agarose powder in an
appropriate buffer, such as TAE or TBE, to be used in
electrophoresis. The agarose is dispersed in the buffer before
heating it to near-boiling point, but avoid boiling. The melted
agarose is allowed to cool sufficiently before pouring the solution
into a cast as the cast may warp or crack if the agarose solution is
too hot. A comb is placed in the cast to create wells for loading
sample, and the gel should be completely set before use.
The concentration of gel affects the resolution of DNA
separation. For a standard agarose gel electrophoresis, a 0.8%
gives good separation or resolution of large 5-10kb DNA
fragments, while 2% gel gives good resolution for small 0.2-1kb
fragments. 1% gels are common for many applications
Loading of samples
Once the gel has set, the comb is removed, leaving wells where
DNA samples can be loaded. Loading buffer is mixed with the
DNA sample before the mixture is loaded into the wells. The
loading buffer contains a dense compound, which may be
glycerol, sucrose, or Ficoll, that raises the density of the sample
so that the DNA sample may sink to the bottom of the well. If
the DNA sample contains residual ethanol after its preparation,
it may float out of the well. The loading buffer also include
colored dyes such as xylene cyanol and bromophenol blue used
to monitor the progress of the electrophoresis. The DNA
samples are loaded using a micropipette.
Electrophoresis
Agarose gel electrophoresis is most commonly done horizontally
in a submarine mode whereby the slab gel is completely
submerged in buffer during electrophoresis.
For optimal resolution of DNA greater than 2 kb in size in
standard gel electrophoresis, 5 to 8 V/cm is recommended (the
distance in cm refers to the distance between electrodes, therefore
this recommended voltage would be 5 to 8 multiplied by the
distance between the electrodes in cm). Voltage may also be
limited by the fact that it heats the gel and may cause the gel to
melt if it is run at high voltage for a prolonged period, especially if
the gel used is LMP agarose gel.
Too high a voltage may also reduce resolution, as well as
causing band streaking for large DNA molecules. Too low a
voltage may lead to broadening of band for small DNA
fragments due to dispersion and diffusion.
A DNA marker is also run together for the estimation of the
molecular weight of the DNA fragments.
Visualization / Staining
DNA as well as RNA is normally visualized by staining with
ethidium bromide, which intercalates into the major grooves of the
DNA and fluoresces under UV light. The ethidium bromide may be
added to the agarose solution before it gels, or the DNA gel may be
stained later after electrophoresis. Destaining of the gel is not
necessary but may produce better images.
Other methods of staining are available;
examples are SYBR Green, GelRed, methylene blue, brilliant
cresyl blue, Nile bluesulphate, and crystal violet. SYBR Green,
GelRed and other similar commercial products are sold as safer
alternatives to ethidium bromide as it has been shown to be
mutagenic in Ames test, although the carcinogenicity of ethidium
bromide has not actually been established. SYBR Green requires
the use of a blue-light transilluminator. DNA stained with crystal
violet can be viewed under natural light without the use of a UV
transilluminator which is an advantage; however it may not
produce a strong band.
When stained with ethidium bromide, the gel is viewed with
an ultraviolet (UV) transilluminator.
Standard transilluminators use wavelengths of 302/312-nm
UV-B. The transilluminator apparatus may also contain
image capture devices, such as a digital or Polaroid camera
that allow an image of the gel to be taken or printed.
Applications
Estimation of the size of DNA molecules following
restriction enzyme digestion, e.g. in restriction mapping of
cloned DNA.
Analysis of PCR products, e.g. in molecular genetic
diagnosis or genetic fingerprinting
Separation of restricted genomic DNA prior to Southern
transfer or of RNA prior to Northern transfer.
Agarose gel electrophoresis is a method of gel
electrophoresis used in biochemistry, molecular biology,
and clinical chemistry to separate a mixed population of
DNA or proteins in a matrix of agarose.
The proteins may be separated by charge and/or size
(IEF agarose, essentially size independent), and the DNA
and RNA fragments by length.
Biomolecules are separated by applying an electric field
to move the charged molecules through an agarose matrix,
and the biomolecules are separated by size in the agarose
gel matrix.
V. MAGENDIRA MANI
ASSISTANT PROFESSOR
PG & RESEARCH DEPARTMENT OF BIOCHEMISTRY
ISLAMIAH COLLEGE (AUTONOMOUS)
VANIYAMBADI
https://tvuni.academia.edu/mvinayagam