Electrophoresis class

2. 1. Definitions
2. Theory of Electrophoresis
3. Types of Electrophoresis
4. Electrophoretic Technique
5. General Procedures
6. Application of Electrophoresis.

3. History
Father of Electrophoresis
Arne Tiselius
The Nobel Prize in Chemistry 1948
"for his research on electrophoresis and
adsorption analysis, especially for his
discoveries concerning the complex nature
of the serum proteins"

4. What is Electrophoresis?
⚫ A method used in biochemistry and
molecular biology to separate charged
particles such as, proteins and nucleic
acids.
⚫ This is achieved by moving negatively/
positively charged molecules through an
support medium under an electric field.

5. Theory of Electrophoresis
⚫ Many biological molecules have ionisable groups
eg. proteins, nucleic acids
⚫ In Solution with pH < pI
-> ampholyte has overall +ve charge
⚫ In Solution with pH > pI
-> ampholyte has overall –ve charge
⚫ Under an electric field
-> cations migrate to cathode
-> anions migrate to anode.

6. ⚫ Principle of Electrophoresis is based on,
Movement of charged particles through an
supporting medium when subjected to an
electric field.
Principle of Electrophoresis

7. ⚫ Rate of migration depends on:
➢ Net electrical charge of molecule
➢ Size & shape of molecule
➢ Electric field strength
➢ Properties of supporting medium
➢ Temperature of operation
Factor affecting Rate of migration

8. TYPES OF ELECTROPHORESIS
⚫ Based on the mode of operation and
separation, electrophoresis is classified
into……

9. Instrumentation and Reagents
⚫ Electrophoretic tank
⚫ Power pack
⚫ Support medium
⚫ Buffer
⚫ Staining
⚫ Detection and quantification

10. Casting tray
Gel combs
Power supply→
Gel tank→ Cover
Electrical leads

Electrophoresis Equipment

11. ⚫ Electrophoretic tank – Tank is mead up
of two chambers (boxes) which hold the
buffer and electrophoretic support on
which separation occures.
⚫ Each box contains an electrode mead up
of carbon or platinum.
⚫ Power pack- The function of power pack
in electrophoretic process is to supply
electrical power.

12. ⚫ Buffer – The buffer serves as a multifunctional
component in the electrophoretic process as it……
⚫ Carries the applied current
⚫ Maintain the pH (8.6) during process
⚫ Determines the electrical charge & extent of
ionization.
⚫ Barbital buffers & Tris-boric acid-EDTA
buffers.
Buffer

13. ⚫ The support medium provides the matrix in which
separation takes place. Various types of support
media are used in electrophoresis.
⚫ Agarose gel
⚫ Cellulose acetate
⚫ PAGE
⚫ paper
Support medium

14. ⚫ To visualize/locate separated protein fractions.
⚫ The commonly used stains are……
⚫ Amido Black B and Coomassie Brilliant for Serum
proteins
⚫ Ethidium bromide for DNA fragment
⚫ Silver nitrate for CSF proteins.
Staining

15. ⚫ Once electrophoretic separation and staining are
complete, it possible to quantify the individual
zones either as % of each fraction present or
absolute conc. by densitometry.
Electrophoretic strip moved on an optical system,
absorbance of each fraction displayed on recorder
chart.
Detection and Quantification

16. Separation of
plasma proteins
by
Agarose Gel Electrophoresis

17. Technique (Process) of Gel electrophoresis
⚫ Gel preparation
⚫ Sample application
⚫ Adjustment of voltage or current - DIRECT
CURRENT ! (gel-electrophoresis about 70 - 100 volts)
⚫ Separation time: minutes
(e.g. gel-electrophoresis of serum proteins 30 min.)
⚫ Electrophoresis in supporting medium.
⚫ fixation, staining and destaining
⚫ Evaluation:
➢ qualitative (standards)
➢ quantitative (densitometry)

18. Agarose is a linear polymer extracted from seaweed.
Agarose
D-galactose 3,6-anhydro
L-galactose
Making the gel
(Gel preparation)

19. An agarose gel is prepared by
combining agarose powder
and a buffer solution.
Agarose
Buffer
Flask for boiling

20. Gel casting tray & combs

21. Seal the edges of the casting tray and put in the combs. Place the
casting tray on a level surface. None of the gel combs should be
touching the surface of the casting tray.
Preparing the Casting Tray

22. Agarose Buffer Solution
Combine the agarose powder and buffer solution. Use a
flask that is several times larger than the volume of buffer.

23. The agarose solution is boiled
until clear.
Gently swirl the solution periodically when heating
to allow all the grains of agarose to dissolve.
Melting the Agarose
Agarose is insoluble at room
temperature .

24. Allow the agarose solution to cool slightly (~60ºC) and then
carefully pour the melted agarose solution into the casting
tray. Avoid air bubbles.
Pouring the gel

25. Each of the gel combs should be submerged in the melted
agarose solution.

26. When cooled, the agarose polymerizes, forming a flexible gel. It should
appear lighter in color when completely cooled (30-45 minutes).
Carefully remove the combs and tape.

27. Place the gel in the electrophoresis chamber.

28. buffer ➔
Add enough electrophoresis buffer to cover the gel to a
depth of at least 1 mm. Make sure each well is filled
with buffer.
Cathode
(negative)
Anode
(positive)

wells
  

29. Bromophenol Blue
(for color) :
Sample Preparation
Mix the samples of protein with the tracking dye.
This allows the samples to be seen when loading onto the gel.

30. Loading the Gel
Carefully place the pipette tip over a well and gently expel
the sample. The sample should sink into the well. Be careful
not to puncture the gel with the pipette tip.

31. Place the cover on the electrophoresis chamber, connecting the
electrical leads. Connect the electrical leads to the power supply. Be
sure the leads are attached correctly - proteins migrates toward the
anode (red). When the power is turned on, bubbles should form on the
electrodes in the electrophoresis chamber.
Running the Gel

35. ⚫ Fixation –
⚫ After completion of electrophoresis, the
gel (supporting medium) is placed in a
fixative (7% acetic acid).
⚫ This prevent the diffusion of separated
proteins.

36. Evaluation of separated protein fractions
Densitometry
Densitometer is used for scanning of separated proteins in the
gel. Scanning the pattern gives a quantitative information about
protein fractions.

37. Point of application

38. Application of Electrophoresis
⚫ Electrophoretic technique is used to…
⚫ Separate plasma proteins, CSF proteins and
proteins in other biological fluids to find out
various disease conditions.
⚫ It also used to separate DNA.
⚫ Helps in the separation of different Hb and
diagnosis of Hemoglobinopathies.
⚫ It is used to separate isoenzymes.
⚫ It is used to separate lipoprotein and to study
different lipoproteinemias.

39. The use of protein electrophoresis in
diagnostics of diseases
Electrophoretic patern is constant under
physiological conditions (intensity of bands).
Spectrum of plasma proteins changes under
various diseases (their ratio)
evaluation of electrophoretic patern
(bands or peaks)

40. Serum protein electrophoresis – agarose gel
Serum proteins (6.0 – 8.0 g/dL)
are separated into 6 groups:
Albumin (3.5 – 5.0 g/dL)
α1 – globulins (0.1 – 0.4 g/dL)
α2 – globulins (0.4 – 1.3 g/dL)
β1 – globulins & (0.6 – 1.3 g/dL)
β2 - globulins
γ – globulins (0.6 – 1.5 g/dL)

42. Serum proteins electrophoresis in diagnostics
of diseases
Normal pattern
Reference ranges:
Total protein 6.0 – 8.0 g/dL
Albumin 3.5 – 5.0 g/dL
α1-globulins 0.1 – 0.4 g/dL
α2-globulins 0.4 – 1.3 g/dL
β-globulins 0.6 – 1.3 g/dL
γ-globulins 0.6 – 1.5 g/dL

44. Acute inflammatory response
• Immediate response occurs
with stress or inflammation
caused by infection, injury or
surgical trauma
• normal or ↓ albumin
• ↑ α1 and α2 globulins
α1 α2-globulins

46. Chronic inflammatory response
• Late response is correlated
with chronic infection
(autoimmune diseases, chronic
liver disease, chronic infection,
cancer)
• normal or ↓ albumin
•↑α1 or α2 globulins
•↑↑ γ globulins
Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM
α1 α2 γ-globulins

48. Liver damage - Cirrhosis
• Cirrhosis can be caused by
chronic alcohol abuse or viral
hepatitis
• ↓ albumin
• ↓ α1, α2 and β globulins
• ↑ Ig A in γ-fraction
Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM
γ-globulins

50. Hepatic cirrhosis
M. Zaharna Clin. Chem. 2009
Decreased albumin (synthesis)
Increased gamma globulins
(polyclonal gammopathy)
Albumin 1 2  
“- bridging”
50

52. Nephrotic syndrome
• the kidney damage illustrates the
long term loss of lower molecular
weight proteins
(↓ albumin and IgG – they are
filtered in kidney)
• retention of higher molecular weight
proteins (↑↑ α2-macroglobulin and
↑β-globulin)
α2-globulin β-globulin fractions

53. Nephrosis
Condition Albumin
Globulins
1 2 β γ
Nephrosis N N
Albumin 1 2  
Decreased albumin
Increased 2-macroglobulin
Decreased gamma globulins
53

54. Hypogammaglobulinemia
Albumin 1 2  
Decreased gamma globulins Condition Albumin
Globulins
α1 α 2 β γ
Hypogammaglo
-bulinemia
N N N N
M. Zaharna Clin. Chem. 2009

56. Multiple myeloma (Monoclonal gammopathy)
Multiple myeloma is caused by
monoclonal proliferation of β-lymphocytal
clones. These „altered“ β-cells produce an
abnormal immunoglobulin paraprotein.
Production of paraprotein is associated
with benign monoclonal gammopathy
(leucemia) and multiple myeloma.
Paraproteins can be found in a
different position: between α-2 and
γ-fraction.
a sharp gamma globulin band
(M band)
M band

57. Monoclonal gammopathy
Albumin 1 2  
Albumin decreased
Sharp peak in gamma region

59. $

60. Thank you…..