electrophoresis

2. WHAT IS ELECTROPHORESIS?
 Electrophoresis is motion of dispersed particles
relative to a fluid under the influence of a spatially
uniform electric field.
 This electro kinetic phenomenon was observed for the
first time in 1807 by Reuss.
 There are several types of electrophoresis but the
concept is same.

3. TYPES OF ELECTROPHORESIS
1. HIGH RESOLUTION ELECTROPHORESIS
2. AGAROSE GEL ELECTROPHORESIS
3. POLYACRYLAMIDE GEL ELECTROPHORESIS
4. ISOELECTRIC FOCCUSING
5. ROUTINE ELECTROPHORESIS
6. CAPILLARY ELECTROPHORESIS
7. TWO DIMENSIONAL ELECTROPHORESIS
8. IMMUNOCHEMICAL ELECTROPHORESIS
IMMUNOFIXATION ELECTROPHORESIS
ELECTROIMMUNOASSAY
9. PULSED FIELD ELECTROPHORESIS

4. GENERAL PRINCIPLE
 Electrophoresis is a technique used to separate
and sometimes purify macromolecules -
especially proteins and nucleic acids - that differ
in size, charge or conformation
 When charged molecules are placed in an
electric field, they migrate toward either the
positive or negative pole according to their
charge.
 In contrast to proteins, which can have either a
net positive or net negative charge, nucleic acids
have a consistent negative charge imparted by
their phosphate backbone, and migrate toward
the anode.

5. ROUTINE ELECTROPHORESIS
 Routine electrophoresis is a generic term for the
traditional clinical laboratory electrophoresis which is
performed on a rectangle shaped slab of gel.
 It is mostly used for separation of protiens and has some
use in separating nucleic acids.
 Generally several patient specimen and control can be
placed on one gel and solute separated in one run.
 This electrophoresis is sometimes called as zone
electrophoresis.

6. GEL ELECTROPHORESIS
 ELECTRO= flow of electricity
PHORESIS= to carry across
• A gel is a colloid, a suspension of tiny particles in a
medium, occurring in a solid form, like gelatin.
• This refers to separation of charged particles located in
a gel when an electric current is applied.
• Charged particles include DNA, RNA, amino acids,
peptides etc.

7. Gel electrophoresis apparatus

8. STEPS
1.PREPARE AGAROSE GEL
2. ADD ETHIDIUM BROMIDE
3.POUR INTO CASTING TRAY
4. PUT GEL IN PLASTIC TRAY & ADD BUFFER
5. SAMPLES ARE PLACED IN GEL
6. START ELECTROPHORESIS
7.AFTER ELECTROPHORESIS IS COMPLETE READ UNDER
UV LIGHT

9. FACTORS INFLUENCING
ELECTROPHORESIS
AGAROSE CONCENTRATION higher
concentration of agarose facilitate separation
of smaller DNA.
VOLTAGE higher voltage cause larger DNA
fragments to move larger distance.

10. 1
Plasmid
Vector
DNA
2
2
1
Add DNA Sample onto
agarose gel lane 2
(DNA ladder is in lane 1)
3
NEGATIVE
ELECTRIC
CURRENT
POSITIVE
DNA bands
Are
separated
by size
4
Dye
added
binds to
DNA
5 DNA bands are
exposed on film
Under uv light
DNA is
visualized
1KB
500 bp
200 bp
GEL ELECTROPHORESIS FOR DNA
SEPARATION

11. POLYACRYLAMIDE GEL
ELECTROPHORESIS
 SDS-PAGE ( sodium dodecyl sulfate polyacrylamide gel
electrophoresis)
 widely used in biochemistry, molecular, forensic and
genetics to separate proteins according to their
electrophoretic mobility( a function of length/
molecular weight)
 separates protein only on basis of molecular weight
and no other property.

12. PROCEDURE
TISSUE PREPRATION

13. PREPARING ACRYLAMIDE GEL

14. POURING OF GEL

15. ELECTROPHORESIS

16. STAINING
 COOMASSIE BRILLIANT BLUE MOST COMMONLY USED
DESTAINED WITH METHANOL/ACETIC
ACID.
20-60 MINUTES
 SILVER STAINING IF THE STAIN IS LIGHT.
DEVELOP WITH SODIUM SULPHATE/
FORMALDEHYDE

17. 1
PROTEIN
2
- - - - - - - -
ADD SDS
SDS binds to amino acid residue
And gives uniform negative charge
To protein. With heat protein is
Linearized.
3
Add protein sample on SDS PAGE
gel lane 2
1 2
4
NEGATIVE ELECTRIC
CURRENT
POSITIVE
Protein bands
Are separated by
size
PROTEIN ELECTROPHORESIS METHOD

18. ADVANTAGES
 Polyacrylamide forms gels with pores of a much more controlled and
uniform size than does agarose.
 Consequently, polyacrylamide gels can be used to separate molecules
that differ in size by as little as 2% of their molecular weight.
 The range of pore sizes possible is also much broader.
 Acrylamide is particularly superior when very small pore sizes are
needed. It can be used to effectively separate small molecules (i.e.
DNA oligonucleotides of 100 bases or less)
 Another advantage of acrylamide as a gel matrix is that it is much
stronger than agarose, producing gels that do not tear as nearly as
easily.
 It is also possible to load larger quantities of material onto
acrylamide gels and to recover the material in a very pure form.

19. DISADVANTAGES
 Polyacrylamide gels are difficult to prepare when
compared with agarose gels and the risk associated with
their preparation is much greater.
 It is most dangerous in its powder form, when it is easily
inhaled.
 Masks, gloves and safety glasses must always be worn
when working with polyacrylamide powder.

20. USES
 SDS PAGE:-
MOLECULAR BIOLOGY
BIOCHEMISTRY
GENETIC STUDY
FORENSIC
MICROBIOLOGY
 GEL ELECTROPHORESIS:-
MASS SPECTROMETRY
RFLP
PCR
CLONING
DNA SEQUENCING
SOUTHERN BLOT

21. ELECTROPHORESIS FOR
SICKLE CELL ANAEMIA
Pattern of hemoglobin electrophoresis from several different individuals.
Lanes 1 and 5 are hemoglobin standards. Lane 2 is a normal adult.
Lane 3 is a normal neonate. Lane 4 is a homozygous HbS individual.
Lanes 6 and 8 are heterozygous sickle individuals.
Lane 7 is a SC disease individual.

22. ELECTROPHORESIS FOR
MULTIPLE MYELOMA
The normal plasma protein electrophoretic pattern shows a heavy albumin band
(to the left) and lighter bands which are alpha, beta and gamma mobility
globulins. The serum from the patient with multiple myeloma shows a heavy
band which is the abnormal monoclonal immunoglobulin (referred to as a
paraprotein). Because it is monoclonal (all molecules identical) it is a very
narrow discrete band, which in this patient has gamma mobility.
α β γ

23. LIPOPROTEIN ELECTROPHORESIS
CHYLOMICRON