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Conjugation of Antibody to HRP

Author: Nanci Donacki
Source: Contributed by Nanci Donacki
Date Added: Tue May 14 2002
Date Modified: Wed Apr 28 2004

 Reagents and Materials

  1.  PBS
  2. PBS: 0.05M EDTA
  3. PBS: 0.01% thimerosal
  4. Conjugate Storage Buffer
  5. SATA
  6.  DMF
  7. Hydroxylamine hydrochloride
  8.  NaOH Pellets
  9. PD10 Column or Sephadex G25
  10. Sulfo-SMCC

 Preparation of Thiolated Antibody

  1. Concentrate the antibody to 5-7 mg/ml
  2. Dissolve 10 mg of SATA in 1 ml DMF (SATA:DMF)
  3. Add 3.0 æl SATA:DMF per mg of antibody
  4. Gently stir solution for 2 hours at room temperature.
  5. Dissolve 0.5 g hydroxylamine hydrochloride in 10 ml PBS:EDTA
  6. Add 0.25 g NaOH pellets to this solution to neutralize to approximately pH 7.0
  7. Add to antibody solution at 6.49 æl/æl SATA.
  8. Gently stir for 30 minutes at room temperature.
  9. Equilibrate column with PBS:EDTA.
  10. Desalt thiolated antibody on column.  Collect in the smallest volume possible without collecting any SATA.  Allow free SATA (2nd peak) to come off the column.
  11. Store the thiolated antibody at 2-8oC for no more than one hour.
  12.  Re-equilibrate the column with PBS.

 Preparation of Maleimide:HRP

  1. Weigh out 1.067 mg HRP for each mg of thiolated antibody.
  2. Add 1.1 mg Sulfo-SMCC per mg of HRP
  3. Add PBS to a final concentration of 8 mg/ml HRP
  4. Mix for 2 hours on a rotator.
  5.  Desalt the HRP on PBS equilibrated column.  Collect the first peak.

 Preparation of Ab:HRP Conjugate

  1.  Immediately mix the HRP with the thiolated antibody at 1 mg HRP per mg antibody.
  2. Gently stir for 16-20 hours at 2-8oC.
  3. Dialyze against 3 changed of PBS, changing every 2 hours, 100X the conjugate volume.
  4.  Dilute the conjugate 2-fold in Conjugate Storage Buffer.  Sterile Filter.
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