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Cellulose acetate electrophoresis

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Cellulose acetate electrophoresis

  1. 1. cellulose acetate electrophoresis
  2. 2. Definition  Electro means Electricity  Phoresis means Separation  Separation of serum proteins by the effect of an electric current. INTRODUCTION:  Electrophoresis is a physical method of analysis which involves separation of the compounds that are capable of acquiring electric change in conducting electrodes.
  3. 3. DEFINITION:  Electrophoresis may be defined as the migration of the charged particle through a solution under the influence of an external electrical field. Ions that are suspended between two electrodes tends to travel towards the electrodes that bears opposite charges
  4. 4. TYPES OF ELECTROPHORESIS l) Zone Electrophoresis  Paper Electrophoresis  Gel Electrophoresis  Thin Layer Electrophoresis  Cellulose acetate Electrophoresis 2) Moving Boundary Electrophoresis  Capillary Electrophoresis  Isotachophoresis  Isoelectric Focussing  Immuno Electrophoresis
  5. 5.  The serum protein electrophoresis (spep) test measures specific proteins in the blood to help identify some diseases. Proteins are substances made up of smaller building blocks called amino acids. Proteins carry a positive or a negative electrical charge, and they move in fluid when placed in an electrical field.  Cellulose acetate electrophoresis:- Cellulose acetate is an acetate salt of cellulose produced by treating cotton with acetic acid using sulphuric acid as a catalyst.  Migration takes place on the buffer film on the surface of the cellulose acetate plate or membrane.  Separation of the proteins is primarily by charge. Cellulose a electrophoresis may be used for qualitative identification of variants, but also with elution for quantitation of the hemoglobin's, A2, A, S, D, Lepore, a-chain variants, Hb H and Hb Bart's
  6. 6. CELLULOSE ACETATE ELECTROPHORESIS  It contains 2-3 acetyl groups per glucose unit and its adsorption capacity is less than that of paper.  It gives sharper bands.  Provides a good background or staining glycoprotein  APPLICATION:  Widely used in analysis of clinical and biological protein sample (albumin and globulins).  Alternative to paper electrophoresis.
  7. 7. Reagents and materials 1. Tris-EDTA Boric Acid (TEB) buffer, pH 8.4. Tris hydroxymethyl amino methane (TRIS): 10.2 g Ethylene diamine tetracetic acid (EDTA): 0.6 g Boric Acid 3.2 g Make up to 1 litre with distilled water . 2. Whatman No. 3 chromatography paper 3. Cellulose acetate membranes. 4. HbA2 control
  8. 8.  Equipment 1. Power supply capable to deliver a constant current, 0-80 mA and up to 400 volts 2. An horizontal electrophoresis tank with adjustable bridge gaps and a polarity indicator (eg. Shandon) 3. Roller mixer. 4. A single beam SP6-200 Spectrophotometer Pye Unicam
  9. 9. Method 1. Haemolysate is prepared from whole blood (in K2EDTA) as previously described. 2. The electrophoresis tank is prepared by filling the tank with 900 ml approximately of TEB buffer wicks are cut from Grade No. 3 chromatography paper and were placed along the 22 cm long bridges in the tank. 3. The cellulose acetate membranes are cut in 40xl00 mm each and soaked (shiny side down) in TEB buffer for 5 minutes. Five strips are plotted and placed on the electrophoresis tanks 4. Voltage current is applied at 250 V for 5 minutes to the membranes to equilibrate the membranes with the buffer. 5. The current is turned off and 8-10 Hl haemolysate (10 g/ul) is applied on each membrane at the cathodal end using a capillary tube
  10. 10. 6. Then the voltage is set at 250-300 V working at constant current of 2 mA for each strip 7. The electrophoresis is run for approximately 45 minutes to one hour until there is a clear area between the bands 8. The current is then turned off and the separated HbA2 on the cellulose acetate membrane is cut and immersed in a tube containing 4 ml of distilled water and the HbA in a tube containing 16ml distilled water. If a haemoglobin variant is present then this is cut separately into 4 or 16 ml ed water depending on the quantity of the variant present. Note that a blank is prepared from the same run by cutting a piece of clear cellulose acetate strip that was immersed in 4ml distilled water. 9. The tubes are then placed on a roller mixer for 30 minutes for the haemoglobin elution.
  11. 11. 10 The strips are removed and the tubes are then centrifuged for 10 minutes at 3000 rpm. 11. The absorbance of each hemoglobin is read at 413 nm against the blank on a spectrophotometer. Factors affecting the result are: correct pH, correct concentration of the buffer and the temperature of the buffer, which may be influenced by the voltage or the environmental temperature. It is advisable, especially in hot climates to keep electrophoretic tanks with buffer refrigerated at 4%C. ldeally the method should be conducted at an environmental temperature below 23 oC. It is necessary, therefore, in warm climates to have air conditioning in the laboratory The quality of the carrier membrane must be good and poor quality should be recognised and discarded. The membrane Should be kept moist.
  12. 12.  Although one of the older methods, cellulose acetate electrophoresis still has a number of applications.  In particular it has retained a use in the clinical analysis of serum samples. Cellulose acetate has the advantage over paper in that it is a much more homogeneous medium, with uniform pore size, and does not adsorb proteins in the way that paper does.  There is therefore much less trailing of protein bands and resolution is better, although nothing like as good as that achieved with polyacrylamide gels.
  13. 13.  The method is, however, far simpler to set up and run. Single samples are normally run on cellulose acetate strips (2.5 cm x 12 cm), although multiple samples are frequently run on wider sheets.  The cellulose acetate is first wetted in electro- phoresis buffer (pH8.6 for serum samples) and the sample (1-2 mm3) loaded as a 1 cm wide strip about one-third of the way along the strip.  The ends of the strip make contact with the electrophoresis buffer tanks via a filter paper wick that overlaps the end of the cellulose acetate strip, and electrophoresis is conducted at 6-8 V cm for about 3 Following electrophoresis, the strip is stained for protein (see Section 10.3.7), destained, and the bands visualised.
  14. 14.  A typical serum protein separation shows about six major bands.  However, in many disease states, this serum protein profile changes and a clinician can obtain information concerning the disease state of a patient from the altered pattern.  Although still frequently used for serum analysis, electrophoresis on cellulose acetate is being replaced by the use of agarose gels, which give similar but somewhat better resolution.  A typical example of the analysis of serum on an agarose gel is shown in Fig. 10.10.  Similar patterns are obtained when cellulose acetate is used. Enzymes can easily be detected, in samples electrophoresed on cellulose acetate, by using the zymogram technique.  The cellulose strip is laid on a strip of filter paper soaked in buffer and substrate.
  15. 15.  After an appropriate incubation period, the strips are peeled apart and the paper zymogram treated accordingly to detect enzyme product hence, it is possible to identify the position of the enzyme activity on the original strip.  An alternative approach to detecting and semiquantifying any particular protein on a strip is to treat the strip as the equivalent of a protein blot and to probe for the given protein using primary antihody and then enryme-linked serondary antibody.  Substrate colour development indicates the presence of the particular protein and the amount of colour developed in a given time is a semiquantitative measure of the amount of protein-Thus, for example, large numbers of serum sample can be run on a wide sheet, the sheet probed using antibodies and clevated levels of a particular protein identified in certain samples by increased levels of colour develop ment in these samples.
  16. 16. ADVANTAGE:  No tailing of proteins or hydrophilic materials.  Available in wide range of particle size and layer thickness.  Give sharp bands and offer good resolution.  High voltage can be applied which will enhance the resolution. DISADVANTAGE:  Expensive  Presence of sulphonic and carboxylic residue causes induced electroosmosis during electrophoresis.