Practical training A1Serum 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 electrophoresisin the practical training A1

power supply (direct current)

electrophoresis chamber

containers for staining and destaining gel

applicator

Serum protein electrophoresisHydragel – agarose gel

Figure is found at http://www.sebia-usa.com/products/proteinBeta.html#

Serum proteins are separated into 6 groups: Albuminα1 - globulinsα2 - globulinsβ1 - globulinsβ2 - globulinsγ - globulins

Hydragel 15/30

• Gels with 15 or 30 wells (serum samples) are used in laboratories of clinical biochemistry.

• Electrophoresis is also used for separation ofisoenzymes,nucleic acids and immunoglobulins

Figure is found at http://www.sebia-usa.com/products/proteinBeta.html#

Hydragel 15/30

Hypergamma Control Pictured

16-30

Figure is found at http://www.sebia-usa.com/products/proteinControl.html

Normal Control Pictured 1-15

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

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

Reference ranges:

Total protein 6.0 – 8.0 g/dLAlbumin 3.5 – 5.0 g/dLα1-globulins 0.1 – 0.4 g/dLα2-globulins 0.4 – 1.3 g/dLβ-globulins 0.6 – 1.3 g/dLγ-globulins 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

• 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

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)

• retention of higher molecular weight proteins (↑↑ α2-macroglobulin and ↑β-globulin)

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

α2-globulin β-globulin fractions

Monoclonal gammopathyMonoclonal gammapathy is caused by monoclonal proliferation of β-lymphocytal clones. These „altered“ β-cells produce an

abnormal immunoglobulin paraprotein.

Production of paraprotein is associatedwith benign monoclonal gammopathy (leucemia) and multiple myeloma.

Paraproteins can be found in a different position: between α-2 andγ-fraction.

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

a sharp gamma globulin band