ELECTROPHORESIS

Electrophoresis is a technique for the analysis and separation

of colloids, based on the travel of colloidal ions under the
influence of an electric field. It is used for the separation of
charged molecules - proteins, DNA and RNA Positive
colloidal particles will travel to the cathode (negatively
charged electrode) and negative to the anode (positively
charged electrode).
The rate of migration will depend on the field strength,
particle charge, and other factors, such as the size and shape
of the colloidal particle. Larger (heavier) particles will travel
slower, while smaller ones will travel faste
• The most applicable type of electrophoresis is zone electrophoresis.
The sample is applied in the form of a dot or tape on the most
homogeneous and inert solid or gelatinous medium such as gel or
paper, an electric field is applied and the molecules travel through the
medium or on its surface for some time during which they divide into
discrete zones (bands, strips).
• It is important to note that the molecules travel through the
electrophoresis buffer, so that the real role of the medium is
supportive, ie. preventing conventional solvent movements caused by
changes in sample temperature and concentration.
• In addition to the above, the medium can have precisely defined pore
sizes and act as a molecular sieve and facilitate or hinder the
separation of molecules.
Paper electrophoresis - filter paper and cellulose acetate
Gel electrophoresis is the most commonly used
electrophoretic technique. The gels used in these separations,
polyacrylamide and agarose, form a three-dimensional
crosslinked structure with a pore size of molecular dimensions
that can be defined. The separation of molecules in these gels
is done on the basis of electrophoretic mobility and size of
molecules (molecular mass).
• Agarose gels (polysaccharide) are used for the separation of large
biomolecules with a molecular weight of 200,000 and more, corresponding to
a pore size above 10 nm in diameter. The most commonly used agarose gels
contain 1 to 0.16% agarose in their composition corresponding to a pore size
of 500 to 150 nm in diameter. These gels are most commonly used to separate
nucleic acids.
• Polyacrylamide gels are used for separations of biomolecules with a
molecular weight of several 1000 to 200 000. Chemical polymerization of
acrylamide monomers with the most common N, N`-methylene-bisacrylamide
results in a three-dimensional network structure (transparent gel). It is
important to emphasize that solutions for the preparation of these gels must be
deaerated, since oxygen interferes with polymerization.
Prikaz pora poliakrilamidnog gela
KADA
NOSAČ

PUFER
MOST
UZORAK
 ―|ı―
140 - 200V

POKLOPAC SA
30min - 2h
ELEKTRODAMA

e-
+ ― ―|ı―
140 - 200V
30min - 2h

e-
 SREDSTVO
FIKSATOR BOJA ZA
ISPIRANJE
T – 55-60 ºC
 DENZITOMETAR

%
55
ALBUMIN

4,8
α1 α2
10 β 
9,2
21
 There are two basic fractions of
serum proteins: - albumin and
globulins

Globulins are divided into three fractions: α, β and γ globulins. The α

globulin strip contains two parts: α1 globulins - α1-antitrypsin, α1-acid
glycoprotein. α2 globulins - haptoglobin, α2-macroglobulin, α2-antiplasmin,
ceruloplasmin. The β globulin band contains transferrin, LDL, complement
proteins The γ globulin band contains immunoglobulins (IgA, IgD, IgE, IgG
and IgM). Paraproteins that occur in multiple myeloma appear in the γ
globulin band.
Acute reaction A ↓, α2 ↑, present in early acute infection,
myocardial infarction, tissue necrosis and in sres
Acute phase reactants (APR): Proteins that respond by increasing or decreasing
clinical conditions in which there is an acute inflammatory response (trauma,
burns, myocardial infarction and exacerbations of inflammatory joint disease.)
Typical prospects are α1- and α2-globulins from α1-AT, α1 -acid glycoprotein and
haptoglobin. In malignant and chronic inflammatory conditions, the sample may
be α2 globulins and γ globulins. There is also an increase in C-reactive protein
(CRP) and a series of complements, which must be measured separately
Nephrotic syndrome:
The picture seen in nephrosis, particularly the Acute
Glomerulonephritides, with decreased albumin and
increased alpha2 -globulins.
Albumin: The concentration reflects the functional capacity of the liver. (Note
that the half-life of serum albumin is 20 days.) γ globulins: polyclonal increase
is often seen in cirrhosis, and fusion of β and γ zones (due to increased IgA) is
often seen in alcoholic cirrhosis. Other serum proteins: α1-antitrypsin -
increased in cirrhosis. Ceruloplasmin - increased in Wilson's disease. α-
fetoprotein - increased in hepatocellular Ca.
Monoclonal gammapathy: disorders of immunoglobulin synthesis consisting of
proliferation of B cell clones. This results in a single homogeneous spike (M
protein) in the β-γ region. This is usually accompanied by a decrease in normal
immunoglobulins. They are usually associated with a malignant clinical course
(eg. multiple myeloma)
Creatine kinase (CK)

CK is a dimer that has 2 subunits:

B subunit (40kDa)
M subunit (40kDa)

CK electrophoresis CK-3
MM
CK-1 CK-2
BB MB

CK has 3 izoenzymes: CK-BB, CK-MB & CK-MM

 CREATINE KINASE IZOENZYMES

PHYSIOLOGICAL CONDITIONS MYOCARDIAL INFARCTION

CK-3 CK-2
MM CK-3
MB
MM
CK-1 CK-2
CK-1
BB MB
BB

CK ISOENZYMES SEPARATED ELECTROPHORETICALLY, BUT CK-MB IS

MORE OFTEN DETERMINED BY IMMUNOESS (ng/mL)