charged particle under the influence of an electric field.
Many important biological molecules, such as amino acids,
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peptides, proteins, nucleotides and nucleic acids, possessionisable groups and, therefore, at any given pH, exist in
solution as electrically charged species, either cations or
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anions .
Under the influence of an electric field these charged
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particles will migrate either to the cathode or to theanode, depending on the nature of their net charge
The equipment required for electrophoresis consist
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basically of two items, a power pack and anelectrophoresis unit.
Electrophoresis requires supporting media such as agarose
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gel , polyacrylamide gel, cellulose acetate paper etc.
Different buffers can be used and are selected depending
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on the samples to be analysed.Buffers like tris borate, barbitone, tris-acetate can be used
for performing electrophoresis.
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SEPARATION OF SERUM PROTEINS BY
AGAROSE GEL ELECTROPHORESIS
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Reagents1. Tris- Borate buffer
Ionic strength- 0.1 M, pH- 8.6
2. Dye solution (Amido Schwarz)
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3. Destaining Solution (3% acetic acid)
4. Agarose gel solution (1%)
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5. Alcohol (95%)6. Equipments
PROCEDURE
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1. Fill the electrode vessel of the cabinet with enough buffer.2. Spread 1.3-1.4 ml of hot agarose gel solution on a clean microscopic
slide uniformly and allow it to stand for 2-5 minutes.
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3. Now apply serum on one end of the slide by soaking a small piece of
filter paper in serum mixed with a tracking dye.
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4. Transfer the slide to the electrophoresis cabinet and establishcontact of slides with buffer by means of a filter paper at both ends.
5. Close the lid and adjust the current at 3 milli ampere or 200 mv
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using power pack and leave it for 2 hours
6. Slides are taken out and alcohol is gently poured.
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Let it stand for 30 minutes at room temperature.7. Dry the slide in hot air oven. Avoid overheating as
the gel may crack.
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8. Stain the slide using the pipette. Allow it to stand
for about 5 minutes.
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9. Destain the slide by keeping it in 3% acetic acid andobserve the pattern.
Normal Patterns and Interpretations
In agarose gel electrophoresis, normal serum is
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separated in to 5 bands. Their normal relative
concentrations are given below:
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Albumin- 55-65%Aplha-1-globulin- 2-4%
Alpha-2-globulin- 6-12%
Beta globulin- 8-12%
Gamma globulin- 12-22%
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