Conjugation of Antibody to HRP

Reagents and Materials

 PBS
PBS: 0.05M EDTA
PBS: 0.01 thimerosal
Conjugate Storage Buffer
SATA
 DMF
Hydroxylamine hydrochloride
 NaOH Pellets
PD10 Column or Sephadex G25
Sulfo-SMCC
 Preparation of Thiolated Antibody

Concentrate the antibody to 5-7 mg/ml
Dissolve 10 mg of SATA in 1 ml DMF (SATA:DMF)
Add 3.0 ml SATA:DMF per mg of antibody
Gently stir solution for 2 hours at room temperature.
Dissolve 0.5 g hydroxylamine hydrochloride in 10 ml PBS:EDTA
Add 0.25 g NaOH pellets to this solution to neutralize to approximately pH 7.0
Add to antibody solution at 6.49 ml/ml SATA.
Gently stir for 30 minutes at room temperature.
Equilibrate column with PBS:EDTA.
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.
Store the thiolated antibody at 2-8oC for no more than one hour.
 Re-equilibrate the column with PBS.
 Preparation of Maleimide:HRP

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

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