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Practical training A1 Serum protein electrophoresis

Pavla Balnov

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 1 - globulins 2 - globulins 1 - globulins 2 - globulins - 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 1-globulins 2-globulins -globulins -globulins 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

Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM

Acute inflammatory response


Immediate response occurs with stress or inflammation caused by infection, injury or surgical trauma normal or albumin 1 and 2 globulins
1 2-globulins

Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM

Chronic inflammatory response


1 2 -globulins

Late response is correlated with chronic infection (autoimmune diseases, chronic liver disease, chronic infection, cancer) normal or albumin 1 or 2 globulins globulins

Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM

Liver damage - Cirrhosis


Cirrhosis can be caused by chronic alcohol abuse or viral hepatitis albumin 1, 2 and globulins Ig A in -fraction
-globulins

Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM

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 ( 2-macroglobulin and -globulin)
2-globulin -globulin fractions

Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM

Monoclonal gammopathy
Monoclonal gammapathy is caused by monoclonal proliferation of -lymphocytal clones. These altered -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 -2 and -fraction. a sharp gamma globulin band

Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM