 Gel electrophoresis is a method used to separate macromolecules like DNA, RNA, and proteins based on their size and charge. It uses a gel as a medium for separation under an electric field. Smaller molecules move faster through the gel towards the positive electrode. The separated molecules can then be visualized and identified. Gel electrophoresis is more efficient at separation than paper electrophoresis and allows for separation of a wider range of molecule sizes.

1. PAPER AND GEL
ELCTROPHORESIS
BY: S.PRIYA
PHARM.D IIIYR

2. ELECTROPHORESIS
 It is a type of protein separation method which relies
on protein sizes to segregate the mixture.
 It is one of the highly efficient techniques of analysis and
sole method for separation of proteins for western blot,
RNA studies etc.
 But, on negative side it also time-consuming, expensive
and technical skilled procedure due to which is less
preferred in health care.
 Electrophoresis is similar to other separation techniques
like chromatography but it differs in-terms of the types of
samples analyzed, the method used for separation,
principle used etc.

3. ELECTROPHORESIS & ITS PRINCIPLE
 Electrophoresis is a method of separation where
in charged molecules migrate in differential speeds in an
applied electric field.“
 The charged molecules under the influence of electric
field migrate towards oppositely charged electrodes.
 Those molecules with +ve charge move towards
cathode and -ve molecules move towards Anode.
 The migration is due to charge on the molecules and
potential applied across the electrodes. The sample
under test is placed at one end of the paper near one of
electrodes. When electricity is applied, the molecules
start moving to respective electrodes.

6. PAPER ELECTROPHORESIS
Paper Electrophoresis is one of the zone
electrophoresis.
Principle:
“The charge carried by a molecule depends on the
pH of the medium. Electrophoresis at low voltage is
not usually to separate low molecular weight
compounds because of diffusion, but it is easier to
illustrate the relationship between charge and pH
with amino acids than with proteins (or) other
macromolecules”.

7. EQUIPMENTS:
Filter paper:
Paper of good quality should contain at least 95% α-
cellulose and should have only a very slight adsorption
capacity.
Apparatus:
Power pack provides a stabilized direct current & has controls for both
voltage & current out put, which have an out put of 0 to 500V and 0 to 150mA
are
The Electrophoretic cell
contains the electrodes, buffer reservoirs, a support for paper and a
supporting transparent insulating cover. The electrodes are usually made of
platinum

8. PROCEDURE
 Paper electrophoresis is a techniques which employs
a Whattman filter paper No.1 which is moistened by a
buffer and then connected at two ends to two opposite
charged electrodes.
 Then sample is applied on to one end and let for
separation of components under electric gradients.
After separation, the paper is dried and stained to get
colored bands.
 These colored bands are recognized for the nature of
sample by comparing with the standard. For a sample
of serum, 5 bands of proteins can be separated by
paper electrophoresis.

10.  Sample application:
The sample may be applied as a spot (about 0.5cm
in diameter) or as a narrow uniform streak.
Special devices are available commercially for this p
urpose. The sample can be applied before the paper
has been equilibrated with buffer (or) after it.
After the sample has been applied to the paper and the
paper has been equilibrated with the buffer.
The current is switched on.

11. Frequent observation is necessary to run electrophoretic apparatus.
Overheating can be avoided by placing the entire equipment in the col
d room.
The process does not take longer than two hours.
After 2 hours switched off the power and paper is removed. Once
removed, the paper is dried in hot oven at 1100C.

12. Detection & Quantitative assay:
To identify the unknown electrophorogram, compare the Unknown
electogram with standard
electrogram under standard conditions.Individual compounds are
usually identified by physical properties by the following methods.
 i) Fluorescence:
a) Staining with “Ethidium bromide” and subsequent
visualization of the electrophoreticgram under UV light makes DNA
& RNA fluoresce and thus facilitates their detection.
b) Flourescamine staining is utilized for detecting amino acids, amino
acid derivatives, peptides & proteins.
c) DANSYl chloride may be used in place of fluorescamine.
ii) UV absorption:
Proteins, Peptides & nucleic acids absorb in the range of 260 to
280nm, this property these can be detect.

13.  iii)) Staining:

14. Applications:
Serum analysis for diagnostic purpose is routinely carried about by paper
electrophoresis.
Muscle proteins, egg white proteins, milk proteins & snake, insect venom
analysis done by this technique.

16.  Gel electrophoresis is a method for separation and
analysis of macromolecules (DNA, RNA and proteins) and
their fragments, based on their size and charge.
 It is used in clinical chemistry to separate proteins by
charge and/or size and in biochemistry and molecular
biology .
 Gel electrophoresis uses a gel as an anticonvective
medium and/or sieving medium during electrophoresis,
the movement of a charged particle in an electrical field.

18.  The separation is more efficient than paper type as
the rate of running of molecules is slow and area of
separation is larger by thickness.
 The sample is applied and subjected to electric field
which can lead to separation of molecules. These
molecules form bands and can be recognized by
staining and comparing with standard sample bands.
 The method is more effective than paper and for
instance from serum sample, 15 proteins bands can
be isolated.

19. TYPES OF GEL:
AGAROSE
• Used for the separation of proteins that are larger than 200 kDa.
• Samples are also easily recovered.
POLYACRYLAMIDE
• Polyacrylamide gel electrophoresis (PAGE) is used for separating
proteins ranging in size from 5 to 2,000 kDa
• Care must be used when creating this type of gel, as acrylamide is
a potent neurotoxin in its liquid and powdered forms.
STARCH
• The gels are slightly more opaque than acrylamide or agarose.
• They are visualised using Napthal Black or Amido Black staining.

20. SEPERATION AND ISOLATION OF DNA
FRAGMENTS:
 The DNA fragments are negatively charged
molecules they can be seperated by forcing them to
move towards the anode under electric field.
 Matrix used is agarose
 The DNA fragments separate according to their size
through sieving effect provided by the gel.
 The seperated DNA fragments can be visualised
only after staining the DNA with a compound known
as ethidium bromide followed by exposure to UV.
(orange bands)
 The seperated bands of DNA are cut out by elution
method.

23.  CBSE Class 12 biology textbook
 Slide share.com
 www.rajaha.com
 biotechniquesden.blogspot.com
 https://en.wikipedia.org/wiki/Gel_electrophoresi
s