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Cellulose Acetate Membrane Electrophoresis

Serum protein electrophoresis. Cellulose Acetate Membrane Electrophoresis. (CAE). xiaoli. - - -. + + +. What is Electrophoresis ?. Its principle is that the charged particles of a sample migrate in an applied electrical field. +. +. +. +. -. cathode. anode.

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Cellulose Acetate Membrane Electrophoresis

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  1. Serum protein electrophoresis Cellulose Acetate Membrane Electrophoresis (CAE) xiaoli

  2. - - - + + + What is Electrophoresis ? Its principle is that the charged particles of a sample migrate in an applied electrical field. + + + + - cathode anode

  3. It is applied for the separation and characterization of proteins, nucleic acids and subcellular-sized particles like viruses and small organelles. Electrophoresis is an analytical method frequently used in molecular biology and medicine.

  4. - - - + + + What is function of Electrophoresis ? Analysis at the level of macromolecules often involves isolation and purification of a particular one from among hundreds or thousands, assaying specifically for this molecule, determining size (molecular weight), shape, and chemical structure. Electrophoresis is used to identify and study charged molecules and macromolecules. + + + + + +

  5. _ electrophoretic mobility (v) _ The electrophoretic mobility v of a molecule is defined as the migration per unit field strength: v = v/x =q /6πη r _ molecule of charge q; electric field of strength x; medium of viscosityη; a spherical molecule of radius r ; a spherical molecule of radius r velocity v The migration of length per time: v : cm / min

  6. - - - + + + Migration velocity depends on • Strength of electric field (Heat) • net charge on molecule (pH) • size and shape of molecules (choice of support) • Properties of supporting medium (viscosity, electroendosmosis)

  7. Electrophoresis of supporting medium • Sodium dodesyl sulphate-plyacrylamide gel electrophoresis (SDS-PAGE) • Agarose gel electrophoresis • Cellulose acetate membrane electrophoresis • Filter paper • Native gels

  8. Barbitone buffer (pH 8.60, 0.06mol/L) pI=4.9 pI=5.28 pI=5.82 pI=7.3 pI=6.8 Albumin α1-globumin α2-globumin β-globumin γ-globumin

  9. NH3+ NH3+ NH2 H- C - COOH H- C - COO- H- C - COO- H+ H+ R R R Effect of pH and buffer on protein charge • Proteins are amphoteric compounds and are therefore either positively or negatively charged • Isoelectric Point - pH where there is no net charge in molecule. PI=pH pI<pH PI>pH

  10. SPECIMENS & MATERIALS 1. Horizontal electrophoresis apparatus 2. Cellulose acetate strips 3. Serum 4. serum applicator 5. Power pack 6. Barbitone buffer (pH 8.60, 0.06mol/L) 7. Whatman 3 MM paper 8. Citrate buffer (pH 6.8, 0.1 mol/L) 9. Amino black 10B stain (0.1%) 10. Washing solution (Methanol: acetic acid: water = 9:1:10)

  11. PROCEDURE • Preparation of Apparatus: • Add the buffer into the chambers. Use filter paper as salt bridge whose one end immerse in the buffer and another put onto the plastic bracket after the buffer in the different (anode’s and cathode’s) chambers have the same height.

  12. 2.Prepare the strip of cellulose acetate frosting varnish

  13. b. Immersing the Strips: Remove the strip of cellulose acetate only with forceps! On the frosting of the strip, lightly mark a beeline at 1.5cm of one end with a pencil. Then moisten a strip by placing it on the surface of the buffer in a flat dish and allow the buffer to soak from below. Immerse the strip completely by gently rocking the dish (about 10 -20 minutes).

  14. 3. Sample Applying: Remove the strip and put between two pieces of filter paper carefully, obsorbe excess buffer solution. Fit the applicator to the block and gently depress, hold down for 3 seconds to completely fill the slots, and release the applicator slowly.

  15. 4. Balancing and Electrophoresis:

  16. Barbitone buffer (pH 8.60, 0.06mol/L) Albumin pI=4.9 α1-globumin pI=5.28 α2-globumin pI=5.82 β-globumin pI=6.8 γ-globumin pI=7.3

  17. 5. Staining: When electrophoresis is completed, turn off the power and disconnect the cords BEFORE removing the chamber cover. Remove the strips, and stain with for 5-10 min in amino black 10 B.

  18. 6. Washing and Observing: Excessive dye from the strip is released by washing repeatedly. Rinse in washing solution for three separate rinses of at least 2 min each with mild agitation. Strips may be stored in 5% acetic acid in the refrigerator if necessary. Strips may be preserved for a report by blotting, air drying and taping to a report form with transparent tape. Washing solution (Methanol: acetic acid: water = 9:1:10)

  19. 1=albumin； 2，3，4，5is α1-，α2-，β- andγ-globumin； 6is origine

  20. a1 a2 g A b a1 a2 g A b Clinic significance (a) 1. Normal value: A 62％～71％ a1 3％～4％ a2 6％～10％ b 7％～11％ g 9％～18％ After scanning (b)

  21. hepatocirrhosis Normal hepatocirrhosis

  22. medullarytumour Normal nephrotic syndrome medullarytumour

  23. Acidic and Basic amino acid

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