ELECTROPHORESIS FOR POST GRADUATES DOING BIOCHEMISTRY.....

1. ELECTROPHORESIS

2.  Multiple myeloma
*a sharp distinct M band appears in -globulin fraction*
 Acute infections
*α1 and α2 globulins are increased*
 Nephrotic syndrome
*decreased albumin with sharp and prominent α2
globulin*
 primary immune deficiency
*Diminished -globulin band*
 α1-Anti trypsin deficiency
*diminished α1-globulin band*

3.  Waldenstrom’s macroglobulinemia
 Primary amyloidosis
 Unexplained peripheral neuropathy
 New onset anemia with renal failure
 Hypercalcemia related to malignancy
 Rouleaux formation on peripheral smear
 renal insufficiency with associated serum
protein elevation.

4.  Unexplained pathologic fracture or lytic lesion
on radiograph...
 CNS infections and inflammations
 Dyslipidemias
 Epidemics (micro organism causing epidemic
can be detected using PFGE)
 Drug monitoring using capillary electrophoresis
Other indications in clinical chemistry

5. Electrophoresis is a physical method analysis
which involves separation of the compounds
that are capable of acquiring electric charge in
conducting electrodes.

6. Electrophoresis may be defined as the
migration of the charged particle through a
solution under the influence of an external
electric field.

7. ELECTROPHORESIS
ZONE
ELECTROPHORESIS
MOVING
BOUNDARY
ELECTROPHORESIS

8. ZONE
ELECTROPHORESIS
PAPER
ELECTROPHORESIS
WHATMANN FILTER
PAPER
ELECTROPHORESIS
GEL
ELECTROPHORESIS
CAPILLARY
ELECTROPHORESIS

9. GEL ELECTROPHORESIS
AGAROSE GEL ELECTROPHORESIS
POLYACRYLAMIDE GEL ELECTROPHORESIS
SDS-PAGE
DISC ELECTROPHORESIS
ISOELECTRIC FOCUSSING
IMMUNOELECTROPHORESIS
TWO DIMENSIONAL ELECTROPHORESIS

10. NO SUPPORTING MEDIUM USED FOR MOVING
BOUNDARY ELECTROPHORESIS
Arne Tiselius
(10 August 1902 – 29 October1971)
Swedish biochemist

11.  Basic components of zone
electrophoresis…..
 supporting medium.
 Buffer chamber.
 Power pack and electrode.
 Sample dissolved in buffer tracking dye.
 Perspex cover to encase the entire chamber.

12. Most commonly used buffers in zone electrophoresis.
*ACETATE
*PYRIDINE
*BARBITONE
*TRIS(2-Amino,2-hydroxy methyl,1-3diol)
most commonly used
*CITRATE

13. Barbitone
buffer(pH-8)
• s.Protein
separation
• Poor resolution
• Week buffer
Phosphate
buffer(pH7)
• Enzymes separation
• Low buffering
capacity
• High conductivity
TRIS- BORATE –
EDTA(TBE)
(pH-8)
• Nucleic acid separation
• Good resolution &high
buffering capacity
• Low conductivity

14. TRIS-acetate-
EDTA(pH-8) (TAE)
• Nucleic acid
separation
• High resolution & high
buffering capacity
• Low conductivity
TRIS- glycine
buffer(pH >8)
• Protein separation
• High buffering capacity
• Low conductivity

15. TRIS- GLYCINE buffer
In 600 ml
distilled
water
3.6grams
TRIS
4.6
grams
GLYCINE

16. 1
• Sample is spotted on supporting medium
2
• Medium is placed between electrodes
3
• Power pack is switched on
4
• Voltage applied till dye has travelled approx 2cm from other end
5
• Medium removed and dried
• stained with coomassie brilliant blue
6
• Destained with 5% acetic acid
7
• Bands are visualized and analyzed using densitometer

17. Supporting
medium
Buffer
Electric
current
Size,shape &net
charge of molecule
Heat and
electroendosmosis

19.  Paper electrophoresis is mainly used for separation of
protein(at low voltage), amino acids and nucleotides(at
high voltage).
 Greatest disadvantage of paper electrophoresis is
diffusion, adsorption and electroendosmosis.
 To over come these issues high voltage is applied 10,000
volts that results in good resolution and rapid separation.
 Small molecules like amino acids and nucleotides are
separated by high voltage electrophoresis
Paper electrophoresis

21.  General procedure is similar to that of paper electrophoresis
 Cellulose acetate electrophoresis has replaced paper
electrophoresis
 Better resolution in short time.
 Negligible adsorption and EEO
 Suitable for fractionation of polysaccharides and lipoproteins
 Bands are easily detected due to transparency of medium.
Cellulose acetate Electrophoresis

22.  Separation of DNA using constant electric field is called
constant field gel electrophoresis(CFGE).
 Separation of DNA altering the strength and direction of
electric field between the electrodes is called as pulse
field gel electrophoresis(PGFE)
 PGFE used to separate high molecular weight DNA.
 CFGE is normal agarose gel electrophoresis for
molecular weight up to 500kb.
 PGFE used to separate human chromosome and yeast
chromosome with sharper bands and high resolution.

24.  Polyacrylamide gels are used for protein fractionation in the presence of
SDS(sodium dodecyl sulphate)
 Polyacrylamide could be used in the form of tube gel(for small volume of
samples)
 Vertical slab gel (for large volume of samples)
 Denaturing of protein sample is needed because protein structure is stable with
hydrophobic interaction, hydrogen bond, and disulphide bond.
 Stable structure does not favour electrophoresis and need to disturb its
structure
 Denaturing agents SDS, urea, and beta mercaptoethanol

26. Denatured
sample
loaded
Running
buffer
(TRIS-
GLYCINE)
ph8.8
80volts
current
applied&
allow it till it
reach 5cm
above lower
end
Staining by
coomassie or
silver staining
Destaining
using
methanol,
acetic acid,
water
mixture

27. Determine molecular weight of proteins
To fractionate protein subunits
To assess the purity of proteins samples
Native PAGE with out SDS
 To separate enzymes and isoenzymes

29. Disc electrophoresis(tube electrophoresis)

30.  Tank of 100 ml buffer forms anode above and another tank of
100 ml forms cathode below.
 Six holes of 2mm diameter are drilled1.5 cm apart and 1mm
above the buffer.
 Any protein specimen can be fractioned using this apparatus.
 The order of resolution is prealbumin, albumin , postalbumins
, ceruloplasmin ,transferrin, post transferrin fractions and
gamma- globulins.
 Lipoproteins separation and studies on Isoenzymes were also
carried out using this apparatus.

Disc electrophoresis
(tube electrophoresis)

32.  IEF is a type of gel electrophoresis in which
compounds are separated in a gel with ph gradient ,
based on differences in iso electric point.
 IEF gel has pre selected pH range.
 Compounds with pH less than iso electric pH acquire
negative charge and move towards anode and vice
versa move towards cathode.
 Migration continues till the molecules reach a pH
region in the gel, which is equal to their iso electric
point.
 At that point molecules become stationary and exist
as zwitter ions.
Isoelectric focusing

33.  To determine iso electric point of protein.
 To separate iso enzymes.
 To fractionate proteins with higher resolution.
 To separate all amphoteric substances.
 To study mono,di,tri substituted derivatives of
proteins.

34. In 2D PAGE, the molecules are first separated by IEF
in a gradient gel, and then by SDS PAGE in a second
gel.
2D PAGE IS USED:
*To resolve proteins and enzymes with high resolution
*To assess purity of proteins.

35. Molecules separated
by…
• pH gradient gel
cast on narrow
cylindrical tube
Gel extruded from
tube…
• Incubated in SDS for
30 min to denature
the protein
Placed on second gel
and stacking gel,
voltage applied and
protein sub units are
separated

37.  IEF combines sensitivity of gel electrophoresis
and specificity of immune reaction.
 This technique is used to quantify antigen.

38.  In capillary electrophoresis, separation is done
with in capillary tube under high pressure and
high voltage to give better resolution in short
time

39.  To separate amino acids.
 To separate oligonucleotides and nucleic acids.
 To separate drugs.
 To separate metal ions.
 To separate enantiomers.

40.  Is used for fractionation of both DNA and
protein
 But with different buffers and at different
current and voltages.
 Agarose gel is used as supporting medium
which is a polysaccharide derivative of agar.
 The pores of agarose gel act as molecular
sieve.
 Pore size of gel is inversely proportional to the
initial concentration of agarose
 0.8% of agarose used for DNA of 0.5-10kb.

41. 1% AGAROSE
100MG
WEIGHED AND
TAKEN IN TO
TEST TUBE

43. Place the test tube in boiling water bath for
about 10minutes

44. After taking out from water
bath evenly mixed agarose
gel taken in to pipette…….

48. POUR THE BUFFER IN TO THE
TANK and connect wicks

49. Run power pack for 1.30 hours at 150volts for
better results

50. For a period of 30 minutes
leave the slide in the fixative

51. Can use drier or hot air oven
Dry the slide till the agarose
fixes to slide

52. Made to 100ml (CBB dye)
Glacial
acetic
acid 7ml
Dye
300mg
Ethanol
50ml

53. Leave the slide in the
CBB stain for 30
minutes

55. Normal serum pattern
Nephrotic syndrome

64. Increased albumin Decreased albumin
• dehydration • Chronic cachectic or wasting
diseases
• Chronic infections
• hemorrhage
• burns
• Protein loosing enteropathies
• Impaired liver function
• malnutrition
• Nephrotic syndrome

65. Increased α1 globulins Decreased α1 globulins
pregnancy α 1 antitrypsin deficiency

66. Increased α2 globulins Decreased α2 globulins
Adrenal insufficiency malnutrition
Adrenocorticosteroid therapy Megaloblastic anaemia
Diabetes mellitus Protein losing enteropathy
Nephrotic syndrome Severe liver disease
Wilson’s disease

67. increasedβ1 or β2
globulins
decreasedβ1 or β2
globulins
Biliary cirrhosis Protein malnutrition
carcinomas
Cushings disease
Diabetes mellitus
hypothyroidism
Iron deficiency anemia
Malignant hypertension
nephrosis
Poly arteritis
Obstructive jaundice

68. Increased -globulins Decreased -globulins
 amloidosis  Agamma globulinemia
 Chronic infections  hypogammaglobulinemia
 Chronic lymphocytic luekemia
 cirrhosis
 Hodgkin’s diseases
 Malignant lymphoma
 Multiple myeloma
 Connective tissue disorders
 Waldenstrom’s
macroglobulinemia