simple and consie slides about essintials in protein electrophoresis

1. Protein Electrophoresis
By
Dr Abdullah A.Hashish
Lecturer of Clinical Pathology Dept.
SCU-FOM

2. Objectives
• Definitions.
• Basic theory.
• Components.
• Types.
• Applications.

3. Definitions
1. Electrophoresis:
migration of charged particles of any size in a liquid
medium under the influence of an electrical field.
2. Iontophoresis or isotachophoresis:
specifically to the migration of small ions.
3. Zone electrophoresis:
each protein zone is sharply separated from
neighboring zones by a protein-free area.

4. Zone electrophoresis

5. Basic Theory
• Depending on the charge they
carry, ionized solutes move
toward either the cathode
(negative electrode) or the
anode (positive electrode) in an
electrophoresis system.

6. Ampholyte
a molecule that is either positively or
negatively charged and becomes
positively charged in a solution that
is more acidic and migrates toward
the cathode while in a more
alkaline solution, the ampholyte
becomes negatively charged and
migrates toward the anode.

7. How to remember:
•Anions  Anode.
•Cations  Cathode.

8. Factors Affecting the Motility
of Ions in an Electrophoretic System
• Net charge of the molecules.
• Size and shape of the molecules.
• Strength of the electrical field.
• Support medium properties.
• Ionic strength of the buffer.
• Temperature.

9. Components

10. Components
1. Power supply:
1. (Voltage, ampere, time).
2. Constant vs. pulsee-power.
2. Buffer:
1. pH. (application & direction of migration).
2. Ionic strength:
1. rate of migration.
2. sharpness of the zone.
3. High ionic strength  high resolution separation.
3. Commonly used: barbital EDTA or Tri-boric EDTA.

11. Components
3. Support media:
1. Buffer solution in a capillary.
2. Membrane of cellulose acetate.
3. Insoluble gel:
– sheets,
– slabs,
– columns of starch,
– agarose, or
– Polyacrylamide.
4. Electrodes.

12. Detection
• Staining:
–Serum proteins in general  Amido Black,
Coomassie Brilliant Blue, Ponceau S.
– Isoenzymes  Nitrotetrazolium Blue.
– Lipoprotein  Fat Red 7B, Oil Red O, Sudan
Black B.
–DNA fragments  Ethidium bromide.
– CSF proteins  Silver nitrate.—

17. What is important to know in the
previous table?
1.Electrophoretic regions.
2.Included proteins.
3.Common function of the
protein.

18. Applications
• used to separate proteins in:
–Serum,
–Urine,
–Cerebrospinal fluid (CSF),
–Other physiologic fluids,
–Erythrocytes and,
–Nucleic acids

19. THANK
YOU