Conjugation of Antibody to HRP

 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 ml 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 ml/ml 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 (2^nd peak) to come off the column.
11. Store the thiolated antibody at 2-8^oC 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 2-8^oC.
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.