Download MBBS Electrophoresis Lecture PPT

ELECTROPHORESIS
GENERAL PRINCIPLE

The term electrophoresis describes the migration of a

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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, possess

ionisable 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 the

anode, 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 an

electrophoresis 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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Reagents
1. 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 establish

contact 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 and

observe 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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