Transcript electrophoresis - Ain Shams University

Faculty of Medicine
Ain Shams University
Medical Biochemistry & 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.
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Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
ELECTROPHORESIS FRACTIONATION
Definition
Electrophoresis is the
migration of charged
particles such as proteins
dissolved in a buffer on,
or in, a supporting
medium when an electric
current is passed through
them.
gel electrophoresis
used to separate macromolecules as
proteins and nucleic acids
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Principle of Separation
• According to charge
• According to size
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
(1) According to charge
• When charged
molecules are placed
in an electric field,
they migrate toward
either the positive
(anode) or negative
(cathode) pole
according to their
charge.
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Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
(2) According to size
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Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Instruments and Reagents
• Power supply
• Tank ( Buffer
,Supporting
media)
• Detection and
Quantification
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Electrophoresis
tank
Cathod
e
Paper
support
medium
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Additional accessories may be required for gel electrophoresis as
combs, glass plates with their separators.
paper electrophoresis apparatus
Gel electrophoresis apparatus
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Factors affecting the rate of migration of charged molecules:
• charge
• Size
• shape
• Nature
• Pore size
• intensity
Molecules
Electric
current
Supporting
media
Buffer
• pH
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Electrophoresis pattern of normal serum proteins
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2 3
4
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Alb 1 2 
Densitometer scanning from cellulose acetate strip.
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Interpretation of the results:
Albumin is the fastest protein , forming the largest
band , and is closest to the positive electrode.
The next 4 bands are globulins; alpha 1, apha
2, beta and gamma globulins, respectively.
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Serum Protein Electrophoresis is useful in
detecting: clinical disorders
.
Albumin
 1 – antitrypsin
deficiency
Immunoglobins
Hypoalbuminemia
• Liver disease
• Kidney disease
Hypogammaglobinemia
Hypergammaglobinemia
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Interpretation of results require knowledge of common
patterns. You should always interpret according to the normal
pattern
Normal Pattern
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
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.
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
-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.
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Nephrotic Syndrome
The nephrotic syndroma pattern is characterized by decreases
in all protein fractions except -2. -2- Macroglobulin is
retained by the kidney owing to its large molecular size.
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Liver Cirrhosis
The typical electrophoresis pattern of liver cirrhosis shows a
merging of the  and  fraction, known as the - bridge
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Lab activities
1-You are provided with a protein sample
,estimate total serum protein with
photometer
2-Interpret the results of your
sample according to electrophoresis
diagram
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Calculation of
the results
Determination of the total
serum proteins
concentration
• Total serum proteins (g/dl)
=AT/AS X conc. of
standard-
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.
Faculty of Medicine
Ain Shams University
Medical Biochemistry & Molecular Biology Department
Interpretation of results:
Normal serum protein electrophoresis using cellulose acetate paper and by elution
gives a pattern in which the various protein fractions as follows:
Protein Fraction
Percentage (%)
Absolute
Concentration (gm/dl)
1- Albumin
55 –65%
3.5 – 5.0
2- 1 Globulins
1.0 –5%
0.17 – 0.33
3- 2 globulins
4.9 –11.2%
0.42 – 0.90
4-  Globulins
7 – 13%
0.52 – 1.05
5-  Globulins
9.8 – 18.2%
0.71 – 1.65