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Immunoprecipitation

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Immunoprecipitation

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Immunoprecipitation

  1. 1. BY KRUPALI AND SHEETAL
  2. 2. CONTENTS <ul><li>PRESIPITATION REACTION </li></ul><ul><li>PROCEDURE </li></ul><ul><li>PRECIPITIN CURVE </li></ul>
  3. 3. <ul><li>TYPES OF IMMUNOELECTROPHORESIS </li></ul><ul><li>Immunodiffusion </li></ul><ul><li>Immunoelectrophoresis </li></ul><ul><li>Countercurrent electrophoresis </li></ul><ul><li>Rocket immunoelectrophoresis </li></ul><ul><li>CROSSED IMMUNOELECTROPHORESIS </li></ul>
  4. 4. Precipitation reaction <ul><li>Antibody and soluble antigen interacting in aqueous solution form a lattice that eventually developed into visible precipitate. </li></ul><ul><li>Antibodies that aggregate soluble antigens are called precipitins. </li></ul>
  5. 6. <ul><li>Precipitate cross linking between antigen and antibody molecules and antigen antibody complex takes place these grow into increasing the large entities that finally precipitate out due to their share size. </li></ul>
  6. 7. <ul><li>Particularly where the antigen antibody interaction of a such a nature that upon the combination a significant number of polar group is masked. The complexes form will have decrease solubility and will tend to aggregate into large insoluble clusters, through contact between their apolar group. </li></ul>
  7. 8. procedure <ul><li>A quantitative precipitation reaction can be performed by placing a constant amount of antibody in a series of tubes and adding increasing amount of antigen to the tubes. </li></ul><ul><li>After the precipitate forms each tube is centrifuged to pellate ,the precipitate is measured. </li></ul><ul><li>Plotting the amount of precipitate against increasing antigen concentration yields a precipitin curve. </li></ul>
  8. 9. Precipitin curve
  9. 11. Radial immunodiffusion <ul><li>Antibody is incorporated into the agar gel as it is poured and different dilutions of the antigen are placed in holes punched into the agar. </li></ul><ul><li>As the antigen diffuses into the gel, it reacts with the antibody and when the equivalence point is reached a ring of precipitation forms. </li></ul>
  10. 12. DOUBLE IMMUNODIFFUSION <ul><li>I n this method both antigen and antibody diffuse radially from wells towards each other thereby establishing of concentration gradient. </li></ul><ul><li>As equivalent is reached the precipitin line forms. </li></ul>
  11. 13. IMMUNOELECTROPHORESIS
  12. 14. <ul><li>A complex mixture of antigens is placed in a well punched out of an agar gel and the antigens are electrophoresed so that the antigen are separated according to their charge. </li></ul><ul><li>After electrophoresis, a trough is cut in the gel and antibodies are added. As the antibodies diffuse into the agar, precipitin lines are produced in the equivalence zone when an antigen/antibody reaction occurs </li></ul>
  13. 15. <ul><li>This test is commonly used for the analysis of components in a patient' serum. </li></ul><ul><li>Serum is placed in the well and antibody to whole serum in the trough. </li></ul><ul><li>This test can also be used to evaluate purity of isolated serum proteins. </li></ul>
  14. 16. COUNTERCURRENT IMMUNOELECTROPHORESIS
  15. 17. <ul><li>In this test the antigen and antibody are placed in wells punched out of an agar gel and the antigen and antibody are electrophoresed into each other where they form a precipitation line . </li></ul><ul><li>This test only works if conditions can be found where the antigen and antibody have opposite charges. </li></ul><ul><li>This test is primarily qualitative, although from the thickness of the band you can get some measure of quantity. </li></ul>
  16. 18. ROCKET ELECTROPHORESIS
  17. 19. <ul><li>A combination of immunoelectrophoresis assay and Mancini assay results in rocket immunoelectrophoresis. </li></ul><ul><li>In rocket immunoelectrophoresis, antigen from wells migrates electrophoretically into an agar gel, which contains specific antiserum. </li></ul><ul><li>This results in rocket shaped precipitate. </li></ul><ul><li>The height of each ‘rocket’ is proportional to the concentration of the antigen in the well. </li></ul>
  18. 20. CROSSED IMMUNOELECTROPHORESIS
  19. 21. <ul><li>In crosses immunoelectrophoresis, protein are first separated by agar gel electrophoresis. </li></ul><ul><li>after which they are electrophorresed into an antibody containing gel at right angles to the direction of the first electrophoresis. </li></ul><ul><li>The technique can be used for analysis of serum proteins. </li></ul>
  20. 22. APPLICATION <ul><li>Determining presence of protein </li></ul><ul><li>Determining the size/molecular weight of a protein </li></ul><ul><li>Monitoring post-translational modification </li></ul><ul><li>Determining protein-protein interaction </li></ul><ul><li>Determining specific enzymatic activity </li></ul>

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