| Conjugation of Antibody to HRP | 点击： 作者： 来源： 时间： 2007-09-13 本站论坛
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Conjugation of Antibody to HRP
Author: Nanci Donacki
Source: Contributed by Nanci Donacki
PBS: 0.05M EDTA
PBS: 0.01% thimerosal
Conjugate Storage Buffer
SATA
Hydroxylamine hydrochloride
PD10 Column or Sephadex G25
Sulfo-SMCC
Preparation of Thiolated Antibody
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Concentrate the antibody to 5-7 mg/ml
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Dissolve 10 mg of SATA in 1 ml DMF (SATA:DMF)
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Add 3.0
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Gently stir solution for 2 hours at room temperature.
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Dissolve 0.5 g hydroxylamine hydrochloride in 10 ml PBS:EDTA
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Add 0.25 g NaOH pellets to this solution to neutralize to approximately pH 7.0
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Add to antibody solution at 6.49
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Gently stir for 30 minutes at room temperature.
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Equilibrate column with PBS:EDTA.
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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.
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Store the thiolated antibody at 2-8oC for no more than one hour.
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Add 1.1 mg Sulfo-SMCC per mg of HRP
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Add PBS to a final concentration of 8 mg/ml HRP
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Mix for 2 hours on a rotator.
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Collect the first peak.
Gently stir for 16-20 hours at 2-8oC.
Dialyze against 3 changed of PBS, changing every 2 hours, 100X the conjugate volume.
Sterile Filter.
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