Practical training A1
Serum protein electrophoresis
Pavla Balínová
Electrophoresis
• Cation = positively charged ion, it moves toward the
cathode (-)
• Anion = negatively charged ion, it moves toward the
anode (+)
• Amphoteric substance = can have a
positive/negative/zero charge, it depends on conditions
Principle:
Some substances have different net charges and can be
separated into several fractions in external electric
field.
But velocity of a particle also depends on the:
size, shape of the particle and given applied voltage
Serum protein electrophoresis on
agarose gel
• Principle:
Serum proteins are negative charged at pH 8.6 (a buffer
helps to maintain a constant pH) and they move toward
the anode at the rate dependent on their net charge.
The separated proteins are fixed and stained by
amidoblack solution.
Serum protein electrophoresis on
agarose gel is a type of horizontal gel
electrophoresis
The figure was found at http://www.mun.ca/biology/desmid/brian/BIOL2250/Week_Three/electro4.jpg
Process of electrophoresis
1. sample application
2. adjustment of voltage or current - DIRECT
CURRENT ! (gel-electrophoresis about 70 - 100 volts)
3. separation time: minutes
(e.g. gel-electrophoresis of serum proteins 30 min.)
4. electrophoresis in supporting medium: fixation,
staining and destaining
5. evaluation:

qualitative (standards)

quantitative (densitometry)
Equipment used for the gel electrophoresis
in the practical training A1
power supply
(direct current)
containers for staining
and destaining gel
electrophoresis
chamber
applicator
Serum protein electrophoresis
Hydragel – agarose gel
Serum proteins are
separated into 6 groups:
Albumin
α1 - globulins
α2 - globulins
β1 - globulins
β2 - globulins
γ - globulins
Figure is found at http://www.sebia-usa.com/products/proteinBeta.html#
Hydragel 15/30
• Gels with 15 or 30 wells
(serum samples) are used
in laboratories of clinical
biochemistry.
• Electrophoresis is also
used for separation of
isoenzymes,nucleic acids
and immunoglobulins
Figure is found at http://www.sebia-usa.com/products/proteinBeta.html#
Hydragel 15/30
Hypergamma Control Pictured
16-30
Normal Control Pictured 1-15
Figure is found at http://www.sebia-usa.com/products/proteinControl.html
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.
Figure is found at http://www.aafg.org
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)
Serum proteins electrophoresis in diagnostics
of diseases
Normal pattern
Reference ranges:
Total protein
Albumin
α1-globulins
α2-globulins
β-globulins
γ-globulins
Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM
6.0 – 8.0 g/dL
3.5 – 5.0 g/dL
0.1 – 0.4 g/dL
0.4 – 1.3 g/dL
0.6 – 1.3 g/dL
0.6 – 1.5 g/dL
Acute inflammatory response
• Immediate response occurs
with stress or inflammation
caused by infection, injury or
surgical trauma
• normal or ↓ albumin
• ↑ α1 and α2 globulins
Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM
α1 α2-globulins
Chronic inflammatory response
α1 α2
• 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
γ-globulins
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
Nephrotic syndrome
• the kidney damage illustrates the
long term loss of lower molecular
weight proteins
(↓ albumin and IgG – they are
filtered in kidney)
α2-globulin β-globulin fractions
• retention of higher molecular weight
proteins (↑↑ α2-macroglobulin and
↑β-globulin)
Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM
Monoclonal gammopathy
Monoclonal gammapathy is caused by
monoclonal proliferation of β-lymphocytal
clones. These „altered“ β-cells produce an
abnormal immunoglobulin paraprotein.
a sharp gamma globulin band
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.
Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM