This document discusses electrophoresis, which is a technique used to separate charged particles like proteins through an electrolyte under the influence of an electric field. It describes how electrophoresis was discovered and some of the major types used like agarose gel electrophoresis. Agarose gel electrophoresis involves preparing an agarose gel on a glass slide, applying a serum sample and dye, running a current through the gel, then fixing, staining and observing the separated protein bands. The document outlines the procedure for agarose gel electrophoresis and lists some factors that affect separation. It also provides examples of how interpreting the results of electrophoresis can help diagnose conditions like multiple myeloma, nephrotic syndrome and cirrhosis.

1. ELECTROPHORESIS
PROF. VIYATPRAJNA ACHARYA
MD, PhD
Dept of Biochemistry, KIMS & PBH, Bhubaneswar

2. ELECTRO + PHORESIS
• Movement of charged particle through an electrolyte
when subjected to an electrical field
• Tiselius (NP-1948)- discovered
• Mixture of charged particles can be separated
• Positively charged particles will move to cathode-cations
• Negatively charged particles will move to anode- anions

3. PROTEINS, LIPOPROTEINS AND HEMOGLOBIN
VARIANTS CAN BE SEPARATED
1. Agarose gel electrophoresis
2. Cellulose acetate electrophoresis
3. Paper electrophoresis
4. Polyacrylamide gel electrophoresis
5. Disc electrophoresis
6. Isoelectric focusing
7. Two-dimensional electrophoresis
8. Capillary electrophoresis
9. Immunoelectrophoresis
Types:
Disc electrophoresis

4. AGAROSE GEL ELECTROPHORESIS
(FOR PLASMA PROTEIN SEPARATION)
Requisites:
1. Electrophoresis chamber
2. Power pack- options of constant voltage and current should be
there
3. Barbitone buffer (pH- 8.6)
4. Agarose powder- 100 ml of agarose powder in 10 ml of buffer
5. Tagging dye- 1 ml buffer saturated with Bromophenol blue
6. Fixative- 70% ethanol
7. Staining solution- 0.5 gm amido black 10B in 100 ml 5% acetic
acid
8. Glass slide
9. Whatman filter paper 3

5. PROCEDURE:
• Take the agarose powder in buffer and boil it while stirring
intermittently till it is fully dissolved. Cool it to 60-700 c and
pipette 1.2-1.4 ml of the gel unto the slide from one side and
allow it to spread evenly. One drop of serum is mixed with the
tagging dye. From one side of the slide the serum is applied with
the help of an applicator or a strip of filter paper. The buffer
tanks are filled with the buffer. Whatman 3 filter paper strips are
cut into pieces same as the width of glass slide. Contact is made
to the buffer and gel plate by means of the filter paper strips.
Direct current is passed for one hour at 150-180 V and 5mA.
Slides were taken out and dipped in fixative solution for 10-15
minutes. Take out the slides and dry them at 700 c till exactly
dry. Then dip the slides in staining solution for 10 minutes and
washed with 5-10% acetic acid and air dry the slides. Observe
the different bands.

6. FACTORS AFFECTING ELECTROPHORESIS
• size of charged particles,
• charge of ions,
• pH of the medium,
• strength of the electric field
• temperature of operation.
• Properties of the supporting medium

7. INTERPRETATION
• In normal electrophoretogram normally 5 bands are seen.
From cathode to anode they are-
• γ-globulin (12%),
• β-globulin (15.5%),
• α-2 globulin (13.5%),
• α-1 globulin (3%)
• albumin (56%)
• Serum electrophoresis helps in diagnosing different disease
conditions. For example, in nephrosis albumin level
decreases and α-2 globulin level increases. In Multiple
myeloma an extra band known as M-band appears
between β and γ bands.

9. CIRRHOSIS

10. NEPHROTIC SYNDROME

11. ACUTE INFLAMMATION

12. MULTIPLE MYELOMA

13. Agammaglobulinaemia

14. CHRONIC HEPATIC DISEASE

15. FAR ADVANCED CIRRHOSIS; ß-Ý FISSION, ↓ALBUMIN

16. FOR MORE PPT ON MEDICAL BIOCHEMISTRY
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