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Peroxidase Conjugation by Periodate Method

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1074

 

Peroxidase Conjugation by Periodate Method

Author: Nanci Donacki
Source: Contributed by Nanci Donacki
 

Reagents

  1. Horseradish peroxidase
  2. Sodium periodate, 0.1M, freshly made
  3. Ethylene glycol
  4. Sephadex G-25
  5. Sodium acetate buffer, 0.001M, pH 4.2
  6. IgG
  7. Carbonate:Bicarbonate Buffer, 1.0 M, pH 9.5
  8. Sodium tetraborate (4 mg/ml) freshly made.
  9.   PBS

  Procedure

  1.   Dissolve 16 mg HRP in 4 ml diH2 O and 0.8 ml of sodium periodate.
  2. Stir for 20 minutes, RT
  3. Add 6 drops ethylene glycol
  4. Stir for 5 minutes, RT
  5.   Filter over a G-25 Sephadex column equilibrated with 1mM acetate
  6. Pool colored fractions and divide into two equal parts.
  7. To the first part, add 14 mg IgG
  8. To the second part, add 10 mg IgG.
  9. To both add 1M carbonate buffer, dropwise, to raise the pH to 9.0-9.5
  10. Stir for 2 hours at room temperature
  11. To both add 0.2 ml sodium tetraborate
  12. Incubate for 2 hours at 2-8o C
  13. Dialyze against PBS.
  14.   Separate on ACA-44 column, eluted with PBS
  15.   Pool fractions and add 10 mg/ml PBS

 

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