Download MBBS Electrophoresis Lecture PPT

Download MBBS (Bachelor of Medicine and Bachelor of Surgery) Latest Electrophoresis Lecture PPT

ELECTROPHORESIS
GENERAL PRINCIPLE

The term electrophoresis describes the migration of a

charged particle under the influence of an electric field.

Many important biological molecules, such as amino acids,

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

anions .

Under the influence of an electric field these charged

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

basically of two items, a power pack and an

electrophoresis unit.

Electrophoresis requires supporting media such as agarose

gel , polyacrylamide gel, cellulose acetate paper etc.

Different buffers can be used and are selected depending

on the samples to be analysed.

Buffers like tris borate, barbitone, tris-acetate can be used

for performing electrophoresis.

SEPARATION OF SERUM PROTEINS BY

AGAROSE GEL ELECTROPHORESIS

Reagents
1. Tris- Borate buffer
Ionic strength- 0.1 M, pH- 8.6

2. Dye solution (Amido Schwarz)

3. Destaining Solution (3% acetic acid)

4. Agarose gel solution (1%)

5. Alcohol (95%)

6. Equipments
PROCEDURE

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.

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.

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

using power pack and leave it for 2 hours

6. Slides are taken out and alcohol is gently poured.

Let it stand for 30 minutes at room temperature.

7. Dry the slide in hot air oven. Avoid overheating as

the gel may crack.

8. Stain the slide using the pipette. Allow it to stand

for about 5 minutes.

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

separated in to 5 bands. Their normal relative

concentrations are given below:

Albumin- 55-65%
Aplha-1-globulin- 2-4%
Alpha-2-globulin- 6-12%
Beta globulin- 8-12%
Gamma globulin- 12-22%