Electrophoresis

Principle and applications of electrophoresis.

2. MOVING
BOUNDARY
ELECTROPHO
RESIS /
FRONTAL
ELECTROPHO
RESIS
pH=7.6
Quantified optically by
change in refractive
index at the boundaries
among these bands
Homogeneous medium

3. pH=8.6

4. Factors affecting rate of migration
Rate of migration (cm/s) per unit field strength (volts/cm)
u = Q
6¶rn

5. Property of supporting media
Cellulose Acetate
Agarose
Polyacrylamide

6. Temperature and
Electrophoresis
•During Electrophoresis
-Denaturation of proteins
-Smile effect
-Heating effect of current and
evaporation
decreases resistance

7. Isoelectric Point

9. Wick Flow
Wick flow is due to flow of
buffer in supporting
medium due to
evaporation of moisture
from gel.
It affect the migration of
sample molecule.

10. Electroendosmosis

11. In case of CSF, the endosmosis is set high to maximize the cathodal
movement of immunoglobulins from the point of application over a span
of the gel that is convenient for visual inspection and detection of
oligoclonal bands of immunoglobulin by optimizing the separation of the
γ-region .

12. IONIC STRENGTH
•Rate of migration is inversely proportion to
ionic strength

13. General Operation
Separation Detection Quantification

14. Lipoproteins
- Sudan Black
Lipoproteins for Oil Red O

15. Plasma Proteins
- Amido black
For plasma protein

16. Hemoglobins
- Coomassie Brilliant Blue Ponceau S for hemoglobins

17. Ethidium Bromide (Fluorescent)


Silver Nitrate for CSF Protein

Sybr Green
(Fluorescent dyes)
for DNA
Sybr Gold
(Fluorescent dyes)
for DNA

19. Paper Electrophoresis
.
Electrophoresis Tank-Inverted V Flynn and Mayo Type.
Hanging strip inverted V type
Covered tank divided in five compartments
/Nylon strip

20. Horizontal strip type
Paper taut in horizontal position
Apparatus is wider than hanging strip type and buffer is farther apart
Heating effect less marked due to smaller volume
of atmosphere.
Covered tank
Whatman Nos.1 and 3MM.(thicker)

21. Shandon Scientific Company New Model
Uses constant voltage
and constant current
supply,either of them
can be selected by
turning the switch.
Uses 50-300 Volts at
maximum 40mA as a
constant current and 3-
40mA at maximum 300
Volts as a constant
voltage

22. Shadon and Evans
Electroselenium Model
Horizontal strip type

23. Sample Applied:Along the pencil line
suitably placed depending on direction of
motion.
In case of protien:Cathodal side of midline
or apex of inverted V
Quantitative Assesment:-Eluting( NaoH,acetic acid,Sodium carbonate)
-Scanning

Duration 8-10hrs .10 microlitre for 3cm ans 20 microlitre for 2 inch strip
Buffer:Barbitone sodium barbitone
(sodium dielthyl barbiturate and di ethyl barbituric acid)
Stain :Bromophenol blue,Bromocresol green ,Lissamine green,Napthaline black

24. Starch Gel Electrophoresis
Molecular
seiving effect
and better
resolution
Connecting
at one end
to a tray
containing
starch gel
and at other
end to
electrode
chambers
Slot former
(1.5x0.1x0.5cm)
6V/cm for 6 hr
and current of
20-30mA.

25. Cellulose Acetate Electrophoresis

26. After staining
strips are made
transparent by
soaking in
whitmore’s oil or
liquid paraffin and
then suitable for
transmittance
densitometry of
the protein bands.
12x2.5 cm
sample applied from
micropipette or
capillary along a line
about 1 cm. on cathode
side of midline
Current:0.4 mA/cm
width of paper.
Dry and suspend in
hot air oven at 80-100
0
C for atleast 10 min.
Cellulose Acetate Electrophoresis
. Its advantage over paper are-
It has better resolving power,speed and ability to be cleared .
No trobule of trailing and background after staining
Washing is almost free from colour.
Excellent seperation of plasma proteins (run-3-4 cm),taking not more than 2 hrs.
Albumin and alpha1 bands are clearly distinguished.
Disadvantage:Costly and greater skill is required to handle the
Strips are brittle and dry.They are difficult to store
10-20 micro litres
6V/cm for 3hr
Stain:PonceauS,Nigrosine

27. Agarose Gel Electrophoresis
Principle and applications
Agarose Gel Electrophoresis

28. Apparatus
Gel Casting Trays
Sample Combs

29. 120V and 7mA
Stain:Amidoswartz 10 B
Destaining agent:Methanol acetic acid

30. Agarose Gel Electrophoresis System
2-3 microlitre using pasteur pipette

31. Polyacrylamide Gel Electrophoresis

32. SDS is an anionic

detergent
The sample is first
boiled for 5min in
buffer containing
•Beta-
Mercaptoethanol
•SDS

33. Seperated by
molecular seiving
effect

34. Apparatus
Glass Plates
Casting Frame
Gel Apparatus
Buffer Tank
Comb
Casting stand gels
with frame

35. Application:
•Serum protein electrophoresis
•Hemoglobin electrophoresis
•Lipoprotein electrophoresis
•Enzymes (zymogen technique)
•ALP isoenzyme
electrophoresis
-Separatin of smaller fragments of DNA
-Separation of nucleic acids

37. •Cirrhosi
Serum protein electrophoresis
in cirrhosis

38. Protein electrophoretic patterns of
serum (Ser)and concentrated urine (Ur)
in a patient with nephrotic syndrome.

42. Serum and urine protein

electrophoretic patterns in a

patient with multiple myeloma.
A predominance
of the
larger complete
immunoglobulin.

A large amount of the
smaller-sized
light chains with only a
small
amount of the
whole
immunoglobulin.

43. Transparent background
Darkly stained fine arcs of antigen
antibodies precipitates
Stain:Amidoswartz 10 B,Nigrosine Stain
Destaining agent:Methanol acetic acid,Ethanol water with trichloroacetic acid
Kappa and Lambda can be obtained by immunodiffusion technique

44. Deficient immunoglobulin synthesis is revealed by a markedly diminished
gamma band.
Effected individuals are prone to recurrent infection

45. A genetic defect causes a deficiency of alpha-1-antitrypsin.

The antiprotease deficiency results in a propensity to develop
emphysema. Since alpha-1-antitrypsin is the major
component of the alpha-1 band, deficiency is
suggested by a reduced alpha-1 band.

Deficiency is confirmed by specific
immunochemical quantification.

46. Bisalbuminemia. Serum protein
electrophoresis with normal pattern in
right lane and bisalbuminemia
consisting of two equal intensity bands
of albumin in left lane. The additional
band of albumin in this patient
migrated more slowly than normal
albumin into the α1-region.

47. 2 Hemoglobin-Haptoglobin
Complex type 1−1
Catalase
(bubbles of O2)
Type 2-2polymers
Hemoglobin
migrated deeply into the gel
no band in the 1-1 position
migrated as a
series of high
molecular weight
polymers, each
binding hemoglobin
Type 1-2
haptoglobin
minor band of 1-1
Series of high
molecular weight
polymers different
from the bands
observed with 2-2. in
middle lane

48. Haemoglobin Electrophoresis
Principle
•At alkaline Ph (8.4-8.6), haemoglobin is a negatively
charged protein and when subjected to electrophoresis
will migrate toward the anode (+).
•Structural variants that have a change in the charge on
the surface of the molecule at alkaline pH will separate
from Hb A.
•Haemoglobin variants that have an amino acid
substitution that is internally sited may not separate and
those that have an amino acid substitution that has no
effect on overall charge will not separate by
• electrophoresis.

49. Note:Test sample should be compared with control sample
containing known normal and abnormal haemoglobin.
Control sample contains Hb A,F,S and C.
Hb A,F,S and are always included in every electrophoresis.
Sample:5 microlitre (Both test and control
sample and covered with 50mm cover slip

50. Cathode (-)
Origin……………………………………………………
………….Carbonic anhydrase
…………..A2'
C…………A2, E, C-Harlem, O-Arab
S…………..D, G, Q-India, Hasharon
…………… Lepore
.……………F
A……………
…………… K-Woolwich
J……………
……………Bart's
N…………..
………….. I
…………... H
Anode (+)
•Schematic representation of relative mobilities of
some abnormal haemoglobins. Cellulose acetate, pH 8.5.

51. Anode (+)
C……………
S………………C-Harlem
……………… Hasharon
Origin…………. O-Arab, Q-India……………...
A………………... D, E, G, Lepore, H, I, N, J
F…………………Bart's, K-Woolwich
Cathode (-)
Schematic representation of relative mobilities of
some abnormal haemoglobins. Agarose gel pH 6.0
using Tri sodium citrate dihydrate buffer using citrate
agar.

52. Haemoglobin electrophoresis pattern
from several different individual.
Lane 1 and 5:Control.
Lane 2:Normal Adult.
Lane 3:Normal Neonate.
Lane 4:Homozygous HbS individual.
Lane 6 and 8:Heterozygous Sickle Cell
individual
Lane 7:Sickle Cell Disease individual.

55. Application:

-Highly sensitive for studying the
microheterogeneity of proteins .
-Useful for separating the isoenzymes.
-Research in enzymology, immunology.
-Forensic, food and agriculture industry.,.

-Human genetic lab

56. Isoelectric Focussing
Electrophoresis
Different gradient of the pH along the length of separating gel.
Ampholyte:low molecular weight and varying isoelectric points

58. Two Dimensional
Electrophoresis
Can separate 1000 to 3000 proteins from
the cell
or
an tissue extract.

59. Schematic representation of 2D Electrophoresis

60. High performance capillary electrophoresis(HPCE) /Capillary zone
electrophoresis (CZE) /Free solution capillary electrophoresis(FSCE)/Capillary
electrophoresis (CE)

61. Sample application is done by either of one method
-High voltage injection
-Pressure injection .

Small amount of sample is required (5-30 μm3).
Molecules are seperated on the basis
of size,hydrophobicity and molecular mass

62. Positively charged molecule reach the cathode first
(electrophoretic migration
+electroosmotic flow).

High voltage

is applied
(up to 50
kV)
The components

migrate at
different
rate along the length.
Although separated by the
electrophoretic
migration,
all the sample is
drawn towards
cathode by
electroendosmosis.
Since this flow is strong, the rate
of electroendosmotic flow is
greater than the
electrophoretic velocity
of the analyte ion, regardless
of the charge.

63. Electro-osmotic flow
DETECTION:
near to cathode
end,
viewing window
- Detected by the
ultraviolet
monitor, transmit
signal and
integrated by
computer.
-Refractive index
-Fluorescence

64. Application:


*Used to separate
-Amino acids
-Peptides
-Proteins
-DNA fragments
-Nucleic acid
-Drugs / even metals
*Multiple myeloma testing (6bands).
*Haemoglobinopathy screening.
*Monitoring chronic alcoholism
(GGT).
*HbA1c

65. Isotachophoresis
•Used for separation of smaller ionic substances.
•They migrate adjacent with contact one another, but not overlapping.
•The sample is not mixed with the buffer prior to run.
•Hence current flow is carried entirely by the sample ions.
•Faster moving ions migrate first and the adjacent ones next with no gap
between the zone .
•All ions migrate at the rate of fastest ion in zones.
•Then it is measured by UV absorbance
Separation of small anions and
cations
Amino acids
Peptides
Nucleotides
Nucleosides
Proteins.

66. Pulsed Field Electrophoresis
•Power is alternately applied to different pair of electrodes/ electrode arrays, so
the electrophoretic field is cycled between two directions.
Electrophoretic field is cycled at 105-1800
Because of which the molecule have to orient to the new field direction .
This permit
separation
of large molecule
like DNA .
Applied:
For typing various
Strain of DNA.