Zone electrophoresis and its types

zone electrophoresis

1. Zone electrophoresis By NIHARIKA SOLA M.PHARM 1 s t sem DEPT. OF PHARMACEUTICAL ANALYSIS

2. Electrophoresis:   Electrophoresis  is a separation technique that is based on the mobility of ions in an electric field.

3. Zone Electrophoresis: Zone electrophoresis (ZE) is an electrophoretic separation technique typically used for analyzing proteins, nucleic acids, and biopolymers. It involves the migration of the charged particle on the stabilizing media. (eg: Paper, Agarose gel, Polyacrylamide gel, Cellulose acetate gel etc.,) Components separated are distributed into discrete zone on the supporting media Supporting media is saturated with the Buffer solution, small volume of sample is applied as narrow bands. On application of sample at the ends of the strip, components migrate at the rate determined by its Electrophoretic mobility. The separated colored components can be separated by Electropherogram.

4. Types of Zone Electrophoresis : Paper Electrophoresis Gel electrophoresis Cellulose acetate Electrophoresis Thin layer electrophoresis

5. 1) Paper Electrophoresis: It is the form of electrophoresis that is carried out using filter paper (Whatmann filter paper) as the stabilizing media. This technique is useful for separation of small charged molecules such as amino acids and small proteins.

6. ADVANTAGES: It is economical Easy to use DISADVANTAGES: Certain compounds such as proteins, hydrophilic molecules cannot be resolved properly due to adsorptive and ionogenic properties of paper which results in tailing and distortion of component bands. Electro osmosis. APPLICATIONS: Serum analysis for diagnostic purpose is done by this technique. Muscle proteins, egg white proteins, milk proteins, snake and insect venom analysis done by this technique. Study of sickle cell disease, haemoglobin C abnormalities.

7. A: normal haemoglobin β chain (HbA) F: normal haemoglobin Y chain (HbF, fetal) S: sickle cell haemoglobin β chain (HbS) C: haemoglobin C β chain (HbC) A2: normal haemoglobin δ chain (HbA2)

8. Serum analysis by electrophoresis

9. 2) Gel Electrophoresis: Separation is brought through molecular sieving technique, based on the molecular size of substances. Gel material acts as “molecular sieve”. Different types of gels which can be used are Agar and Agarose gel, Starch gel, Polyacrylamide gels. Agarose Gel: It is a linear polysaccharide, used as 1% and 3% conc. Pore size is controlled by the % of agarose used. Applications: To separate DNA, proteins, Hb variants, Iso enzymes, etc.,

10. Poly acrylamide Gel Electrophoresis (PAGE) : PAG Electrophoresis is the most widely used method in analysis of complex mixture of proteins. PAGE of proteins can be broadly classified in the following ways:- Homogeneous systems. Multiphasic (discontinuous) systems. SDS PAGE: Sodium dodecyl sulphate – polyacrylamide gel electrophoresis. Most widely used method for analysing protein mixture quantitatively. Useful for monitoring protein purification – as separation of protein is based on the size of the particle. Can also be used for determining the relative molecular mass of a protein.

11. Preparation of poly acrylamide gel: Gather combs, glass plates, spacer (silicone tubing), and binder clips. A comb is used to make wells (lanes) to load samples. Use an appropriate comb depending on the sample size . Thoroughly clean the glass plates with ethanol, and assemble the gel casting mold . Pour acrylamide solution for a separating gel. Overlay with water to prevent contact with air (oxygen), which inhibits polymerization. Allow acrylamide to polymerize for 20-30 minutes to form a gel. Remove the overlaid water . Pour acrylamide solution for a stacking gel; insert a comb and allow the acrylamide to polymerize.

12. Advantages: These gels are stable over wide range of pH and temperature. Gels of different pore size can be formed. Simple and separation speed is good comparatively. Exhibit reasonable mechanical strength . Disadvantages: Gel preparation and casting is time consuming Carcinogenic Complete reproducibility of gel preparation is not possible.

13. ii) Native PAGE: Native gels are run in non-denaturing conditions, so that the analyte’s natural structure is maintained. Separation is based upon charge, size, and shape of macromolecules. It is done by using the polyacrylamide gel(75%). Useful for separation or purification of mixture of proteins. Used up to 3-30% concentration (pH range 4.0 – 9.0). Lower concentration is used for DNA separation and higher concentration is used for the protein separation. SDS is absent in Native PAGE .

14. Applications : -Research tool -Measuring molecular weight -Peptide mapping -Protein identification -Determination of sample purity -Separation of proteins and establishing size -Blotting -Smaller fragments of DNA

15. DNA sampling by gel electrophoresis

16. Blotting techniques: Southern blotting (for DNA) Northern blotting (for RNA) Western blotting (for proteins)

17. Pulsed Field Gel Electrophoresis: Power is applied alternatively to different pair of electrodes. Electrophoretic field is cycled at 105-180°. Because of which the molecule have to orient to the new field direction. This permit separation of large molecule like DNA. Applications: Genome size estimation. DNA fragments obtained by using endonucleases produce a discrete pattern of bands useful for fingerprinting and physical mapping of chromosome. Useful to establish the degree of relatedness among different strains of same species.

18. 3) Cellulose Acetate Electrophoresis: This kind of electrophoresis was developed by Kohn in 1958. Cellulose acetate electrophoresis is more or less very similar to the paper electrophoresis. Biological acetate membrane filter used in this instead of normal chromatography paper . It is much more advantageous than paper because it is a homogeneous system with uniform pore size.

19. Cellulose acetate is an acetate salt of cellulose produced by treating the cotton with acetic acid using sulphuric acid as catalyst. It contains 2-3 acetyl groups per glucose unit and its absorption capacity is less than that of paper. It gives sharper bands. Provides good background or staining glycoprotein . Reagents and materials: Tris-EDTA boric acid buffer of pH 8.4 (for serum samples pH 8.6 buffer is used) Whatmann n0.3 chromatography paper. Cellulose acetate membrane HbA2 control. Equipment: Power supply A horizontal electrophoresis tank with adjustable bridge gaps and polarity indicator. Roller mixer. A single beam spectrophotometer.

20. Procedure: It involves the following steps : First, the cellulose acetate filter paper moistens with the buffer solution. After this, immerse both the ends of cellulose acetate filter paper in a buffer solution. Then sample place at the one end, after which the electric field is applied, that allows migration of molecules to their respective poles depending upon their charge.

21. Applications: - Widely used in the analysis of clinical and biological protein sample (albumin and globulins). - alternative to paper electrophoresis. Advantages: No tailing of proteins or hydrophobic materials. Available in wide range of particle size and layer thickness. Gives sharper bands and good resolution. High voltage can be applied which will enhance the resolution. Disadvantages: Expensive Presence of sulphonic and carboxylic residues causes induced electro osmosis during electrophoresis.

22. 4) Thin Layer Electrophoresis: In 1946, electrophoretic separation of mixture of amino acids and peptides on a thin layer of silica gel. In this electrophoresis studies are carried out in thin layer of silica, keisulghur, alumina. Advantages: Less time consuming. It is advantage over paper electrophoresis. More uniform and finer structure of absorbent is used for thin layer electrophoresis, compared with filter paper ,leads to better resolution. Applications: Widely used in combined electrophoretic-chromatography studies in two dimensional study of proteins and nucleic acid hydrolysates.

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