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Electrophoresis by Anubhav Singh, M.pharmPresentation Transcript
Presented By- Anubhav Singh
M.pharm 1st Sem
IPR, GLA University
Introduction• When a potential difference is applied between the two electrodes in a
colloidal solution, It has been observed that the colloidal particles are carried
to either the positive or negative electrode.
• In other words , they behave as if they are electrically charged w.r.t. the
dispersion medium. This phenomenon is known as electrophoresis.
• Many important biological molecules, such as amino acids, peptides,
proteins, nucleotides and nucleic acids, possess ionisable groups and,
therefore, at any given pH, exist in solution as electrically charged species
either as cations or anions.
• Under the influence of an electric field these charged particles will migrate
either to the cathode or to the anode, depending on the nature of their net
• The common support is a polymer of agarose and acrylamide.
Why to Use Electrophoresis?
• Electrophoresis is a technique used for the separation of biological
molecules based on their movement due to the influence of a direct
• The technique was pioneered in 1937 by the Swedish chemist Arne
Tiselius for the separation of proteins.
• It has now been extended to the separation of many other different
classes of biomolecules including nucleic acids, carbohydrates and amino
• Electrophoresis has become increasingly important in the laboratory for
basic research, biomedical research and in clinical settings for the
diagnosis of disease.
• However, it is valuable as an analytical technique for detecting and
quantifying minute traces of many biomolecules in a mixture.
• It is also useful for determining certain physical properties such as
molecular mass, isoelectric point, and biological activity.
• It is useful to be able to separate the pieces (for recovering particular
pieces of DNA, for forensic work or for sequencing).
Types of ElectrophoresisThere are quite a number of types of electrophoresis commonly used. It is not
possible to go through them all in any detail here, but a brief description of a
few of the most common types follows:
A. SDS Electrophoresis- One of the most common means of analyzing
proteins by electrophoresis is by using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. SDS is a detergent which denatures
proteins by binding to the hydrophobic regions and essentially coating the
linear protein sequence with a set of SDS molecules. The SDS is negatively
charged and thus becomes the dominant charge of the complex. The number of
SDS molecules that bind is simply proportional to the size of the protein.
Therefore the charge to mass ratio should not change with size.
In solution (water), in principle all different sized proteins covered with SDS
would run at about the same mobility. However, the proteins are not run
through water. Instead they are run through an inert polymer, polyacrylamide.
The density and pore size of this polymer can be varied by just how you make it
(concentration of monomer and of cross-linking agent). Thus, the size of
molecules that can pass through the matrix can be varied.
This determines in what molecular weight range the gel will have the highest
B. Native Gels- It is also possible to run protein gels without the SDS.
These are called native gels in that one does not purposely denature the
protein. Here, the native charge on the protein (divided by its mass)
determines how fast the protein will travel and in what direction.
C. Electrofocusing Gels- Another variation of gel electrophoresis is to pour
a gel that purposely has a pH gradient from one end to the other. As the protein
travels through this pH gradient, its various ionizable groups with either pick up
or lose protons. Eventually, it will find a pH where its charge is zero and it will
get stuck (focused) at that point.
E. DNA Agarose Gels- A simple way of separating fairly large fragments
of DNA from one another by size is to use an agarose gel. Agarose is another
type of matrix used for many purposes (such as the support for the growth of
bacteria on plates). DNA does not need a detergent, since it already has a
large under of negative phosphate groups evenly spaced. Thus, as with SDSPAGE, the charge to mass ratio is constant. Also like SDS-PAGE, the
separation results from the matrix itself. The range of size sensitivity can be
varied by changing the density of the agarose.
F. Capillary electrophoresis- It has become popular to separate
molecules electrophoretically by running them into and through a capillary
tube. This is fast and accurate, but does not allow much sample to be loaded
on the gel at once.
Electrophoresis Equipment's And
ProcessA. SAMPLE DELIVERY INSTRUMENTSa. Variable Automatic Micropipettes:
An automatic micropipette is used to deliver accurate, reproducible volumes of
• For the electrophoresis of dyes, load the well with 35-38 microliters of sample.
• Use a clean micropipette tip for loading each sample.
b. Transfer Pipets:
•Disposable plastic transfer pipets can be used, but they are not precise.
Because their volumes cannot be accurately controlled, their use can result in
significant sample waste.
c. Fixed Volume Micropipettes:
•Accurate sample delivery can also be achieved using fixed volume
Micropipettes. These types of Micropipettes are pre-set to a specific volume.
Although the volume can not be changed, these types of Micropipettes operate
similarly to the variable automatic Micropipettes. Most fixed volume pipets do
not have ejector buttons, so the tips must be removed manually.
C. PREPARING THE GEL BED1. Close off the open ends of a clean and dry gel bed (casting tray) by using
rubber dams or tape.
A. Using Rubber dams:
• Place a rubber dam on each end of the bed. Make sure the rubber dam fits firmly
in contact with the sides and bottom of the bed.
B. Taping with labeling or masking tape:
• With 3/4 inch wide tape, extend the tape over the sides and bottom edge of the
• Fold the extended edges of the tape back onto the sides and bottom. Press
contact points firmly to form a good seal.
2. Place a well-former template (comb) in
the middle set of notches. Make sure the
comb sits firmly and evenly across the
CASTING AGAROSE GELS
3. Use a 250 ml flask to prepare the gel solution. Add the following components
to the flask as specified for your experiment
4. Swirl the mixture to disperse clumps of agarose powder.
5. With a marking pen, indicate the level of the solution volume on the
outside of the flask.
6. Heat the mixture to dissolve the agarose powder. The final solution
should appear clear (like water) without any undissolved particles.
i.. Microwave method:
Cover the flask with plastic wrap to minimize evaporation.
•Heat the mixture on High for 1 minute.
•Swirl the mixture and heat on High in bursts of 25 seconds until all the agarose
is completely dissolved.
ii. Hot plate method:
•Cover the flask with aluminum foil to prevent excess evaporation.
•Heat the mixture to boiling over a burner with occasional swirling. Boil until
all the agarose is completely dissolved.
7. Cool the agarose solution to 55°C with
careful swirling to promote even
dissipation of heat. If detectable
evaporation has occurred, add
distilled water to bring the solution up
to the original volume as marked on
the flask in step 5.
8. Seal the interface of the gel bed and tape to prevent the agarose solution from
•Use a transfer pipet to deposit a small amount of cooled agarose to both inside
ends of the bed.
•Wait approximately 1 minute for the agarose to solidify.
9. Pour the cooled agarose solution into the bed. Make sure the bed is on a level
10. Allow the gel to completely solidify. It will become firm and cool to the touch
after approximately 20 minutes.
D. Prepare Gel for Electrophoresis-
E. Sample delivery-
F. Loading of Sample.
G. Running of gel.
H. Result formation•A variety of documentation methods can be used, including drawing a picture of
the gel, taking a photograph, or scanning an image of the gel on a flatbed scanner.
InstrumentVertical Gel Instrument-
Horizontal Gel Electrophoresis
Digital Camera/Gel Documentation System
Tube Gel Units
Slab Gel Units
• Capillary electrophoresis has grown to become a collection of a range of
separation techniques which involve the application of high voltages across
buffer filled capillaries to achieve separations .
• Capillary electrophoresis, then, is the technique of performing electrophoresis
in buffer-filled, narrow-bore capillaries, normally from 25 to 100 pm in
internal diameter (ID).
• A high voltage (typically 10-30 kV) is applied across a narrow bore (25-100
• The capillary is filled with electrolyte solution which conducts current through
the inside of the capillary. The ends of the capillary are dipped into reservoirs
filled with the electrolyte.
• Electrodes (platinum) are inserted into the electrolyte reservoirs to complete
the electrical circuit
• A small volume of sample is moved into one end of the capillary. The capillary
passes through a detector, usually a UV absorbance detector, at the opposite end
of the capillary.
• Application of a voltage causes movement of sample ions towards their
appropriate electrode usually passing through the detector
A plot of detector response with time is generated which is termed an
Two-Dimensional Gel Electrophoresis
• Two-dimensional gel electrophoresis is widely used to separate complex
mixtures of proteins into many more components than is possible in
conventional one-dimensional electrophoresis.
• Each dimension separates proteins according to different properties.
Applications Of Electrophoresis
• DNA Sequencing
• Blotting (DNA Hybridization /Southern Blot)
• Medical Research
• Protein research/purification
• Agricultural testing
• Many others
• Sharma B.K; “ Instrumental Methods of Chemical Analysis”, pp- C-269 – C281.
• Electrophoresis journals: Journal of Separation Science (JSS), Electrophoresis.
• Societies: The American Electrophoresis Society (www.aesociety.org).
• Principles and Practice of Agarose Gel Electrophoresis. The Biotechnology
Education Company, EDVO-Kit- 101.