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