1. Practical training A1 Serum protein electrophoresis Pavla Balínová

2. 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

3. 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.

4. Serum protein electrophoresis on agarose gel is a type of horizontal gel electrophoresis
The figure was found at

5. Process of electrophoresis
1. sample application
2. adjustment of voltage or current - DIRECT CURRENT ! (gel-electrophoresis about 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)

6. Equipment used for the gel electrophoresis in the practical training A1
power supply (direct current)
containers for staining and destaining gel
electrophoresis chamber
applicator

7. 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

8. 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

9. Hydragel 15/30 Hypergamma Control Pictured 16-30
Normal Control Pictured 1-15
Figure is found at

10. 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

11. 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)

12. Serum proteins electrophoresis in diagnostics of diseases Normal pattern
Reference ranges:
Total protein – 8.0 g/dL
Albumin – 5.0 g/dL
α1-globulins – 0.4 g/dL
α2-globulins – 1.3 g/dL
β-globulins – 1.3 g/dL
γ-globulins – 1.5 g/dL
Figure is found at

13. 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
Figure is found at

14. Chronic inflammatory response
α1 α2 γ-globulins
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

15. Liver damage - Cirrhosis
γ-globulins
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

16. 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
Figure is found at

17. Monoclonal gammopathy
Monoclonal gammapathy 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
Figure is found at