Medical Biochemistry and Molecular Biology Department
MEDICAL BIOCHEMISTRY
AND
MOLECULAR BIOLOGY DEPARTMENT
PRACTICAL GUIDE NOTES
ON
ELECTROPHORESIS FRACTIONATION
Medical Biochemistry and Molecular Biology Department
ELECTROPHORESIS FRACTIONATION
ILO of the current topic:
By the end of this topic, the student will be able to: Interpret electrophoresis
results for serum proteins and hemoglobin.
Definition
Electrophoresis is an analytical technique used for the separation of
molecules such as proteins dissolved in a buffer on/or in, a
supporting medium when an electric current is passed through them.
Theory and principle:
Movement of charged molecules in solution upon application of electric field
depends on:
1. Molecular weight
2. Charge
 The positively charged molecules
(cations) migrate towards the negatively
charged electrode (cathode). Likewise,
the negatively charged molecules
(anions) migrate towards the positively
charged electrode (anode).
General requirements for electrophoretic separation include the following:
1- Power supply
2- Tank ( Buffer ,Supporting media)
3-
Detection and Quantification
Electrophoresis
tank
Cathode
Anode
Paper
support
medium
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Additional accessories may be required for gel electrophoresis as combs,
glass plates with their separators,….etc See next figure.
Gel electrophoresis apparatus
Paper electrophoresis apparatus
Factors affecting the rate of migration of charged molecules:
1- Molecular Characteristics
•
234-
5-
The net charge of the molecule, this will depend on the pH of the
medium through which electrophoresis is performed.
• Size and shape of the molecule.
Electrical Field Characteristics: The strength of the electric field.
Buffer properties: pH of the buffer
The nature of the supporting medium e.g. paper electrophoresis
(cellulose acetate) or gel electrophoresis (polyacrylamide gel
electrophoresis) (PAGE).
The temperature during the process of electrophoresis.
Clinical application:
Serum Protein Electrophoresis Using Cellulose Acetate Strips
Electrophoresis is the most common method for analyzing serum proteins. In
clinical laboratories, cellulose acetate is widely used as a supporting
medium.
How does serum protein electrophoresis work?
• In serum protein electrophoresis using a buffer at PH 8.6, the proteins will
carry negative charges (due to the amphoteric nature of the proteins).
When the current is applied, the negatively charged protein particles will
migrate towards the anode (positively charged electrode).
Representative electrophoretic pattern of normal serum proteins and densitometer scanning
Medical Biochemistry and Molecular Biology Department
from cellulose acetate strip
• The individual components of serum proteins will migrate at different rates
depending on their molecular weight, net charges, and isoelectric
points. The isoelectric point of albumin is 4.8, and that of gamma globulins
is about 7.4. At alkaline pH, albumin will have a greater number of negative
charges than the gamma globulin and so will migrate faster towards the
anode.
• As can be seen in the electrophoresis pattern, the slowest gamma
globulins are precipitated near the cathode.
Visualization of proteins
• After proteins are separated by
electrophoresis, they are stained with
Ponceau-S
dye/trichloroacetic
acid
solution and quantitated by densitometry.
The Protein fractions are reported in
grams per volume and as a percentage
from the total serum proteins.
What's in each peak?
Calculation of the results:
A. Determination of the total serum proteins concentration:
Total serum proteins (g/dl) =AT/AS X conc. of standard- Normal value
is 6-8g%
B. Absolute concentration (gm/dl) of protein fractions:
Absolute concentration of a fraction = P/100 X C. Where P = the percent of
each zone and C = the total serum protein concentration in gm/dl. e.g. If p
for albumin is 60%, and total protein is 7 g% so Albumin concentration is
=60/100X7= 4.2 g
Medical Biochemistry and Molecular Biology Department
Interpretation of results: require knowledge of common patterns. As always
you should interpret according to the normal pattern
1-Normal Pattern
Five electrophoretic bands are present in serum protein electrophoresis,
arranged from the anode (positive electrode) towards the cathode (negative
electrode) these bands are: Albumin, 1 globulins, 2 globulins, 
globulins, and  globulins.
Protein Fraction
Percentage
(%)
1- Albumin
55 –65%
Absolute
Concentration
(gm/dl)
3.5 – 5.0
2- 1 Globulins Include: 1
antitrypsin, 1 lipoprotein (HDL),
thyroxin binding globulin.
3- 2 globulins Include: 2
macroglobulin, haptoglobin.
4-  Globulins Include:  lipoproteins
(LDL), fibrinogen
5-  Globulins Include: IgG, IgA, IgM,
IgE, IgD.
1.0 –5%
0.17 – 0.33
4.9 –11.2%
0.42 – 0.90
7 – 13%
0.52 – 1.05
9.8 – 18.2%
0.71 – 1.65
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2-Monoclonal Gammopathy Pattern
The large spike in the gamma region is indicative of an increase in one
immunoglobulin class typically seen in monoclonal gammopathies such as
multiple myeloma.
3--1 Antitrypsin Deficiency
1-antitrypsin deficiency appears as either a decrease or complete absence
of the 1 band depending on whether the condition is heterozygous or
homozygous.
4-Nephrotic Syndrome
The nephrotic syndrome pattern is characterized by decreases in all protein
fractions except -2. -2- Macroglobulin is retained by the kidney owing to its
large molecular size.
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5-Liver Cirrhosis
The typical electrophoresis pattern of liver cirrhosis shows a merging of the 
and  fraction, known as the - bridge
Liver Cirrhosis
Summary SPE Interpretation
Albumin
1 Globulins
2 Globulins
 Globulins
 Globulins
-

-
-
-
-
-
-
-

Hepatic cirrhosis

-
-
-

Nephrotic

-/
-
-/

Pathological state
-1 Antitrypsin
Deficiency
Monoclonal
Gammopathy
Pattern
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Activity: You are provided with a labeled standard and blank to use. Estimate the
concentration of total protein in your given serum sample using the photometer- Then
according to your results calculate the concentration of each fraction in the given
activity diagram. Finally, interpret your results.
Normal Pattern
Interpret this
abnormal pattern
Hemoglobin electrophoresis
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Hemoglobins (Hb) are composed of polypeptide chains called globin,
and iron protoporphyrin heme groups. A specific sequence of amino acids
constitutes each of four polypeptide chains, which are designated , ,
and .Each normal hemoglobin molecule contains one pair of alpha and
one pair of non-alpha chains. In normal adult hemoglobin (Hb A), the nonalpha chains are called beta and the structure is expressed as 2,2 .
Normal blood level for adult men is (14-18 g/dl), while for adult women
is (12-16 g/dl).
Hemoglobinopathies
Are a group of disorders caused either by production of a structurally
abnormal hemoglobin molecule with an altered amino acid sequence (eg.
Hb S which causes sickle cell anemia) or by synthesis of insufficient
quantities of normal hemoglobin (e.g thalassmia).
Hemoglobin electrophoresis:
Electrophoresis is generally considered the best method for separating
and identifying the hemoglobins present in a blood sample.
Procedure:
Initial electrophoresis is performed in an alkaline buffer (pH 8.2-8.6).
Cellulose acetate is the major support medium because it yields rapid
separation of Hb A, F, S and other mutants with minimal preparation time.
However, because of the electophoretic similarity of some hemoglobins, a
procedure that measures some property other than electrical charge may
be needed.
Steps and result:
Are exactly the same as those used in protein electrophoresis, except: the
sample material which is hemolysate of the patient sample, which is
prepared by mixing 1 part of whole blood to 3 parts of hemolysing reagent.
The products of normal and abnormal hemoglobins are shown in the
following figure: