Abstract：
:In this report, we describe a two-step protocol for labeling of an affinity-purified antibody to biotin with horseradish peroxidase (HRP) using cyanuric chloride (CC) as a bridge. The enzyme was first modified with CC, and following chromatography on a PD-10 column, the activated HRP was incubated with the antibody to effect coupling of the two proteins. Assessment of the conjugate product was carried out using ELISA and SDS-PAGE electrophoresis where evidence for high antibody activity, high specific activity of the conjugate preparation, coupling of nearly all the antibody and over 90% of the enzyme was shown. The titer of the conjugate exceeded 1/100,000. High molecular weight complexes were observed in the SDS-PAGE results, indicating an efficient conjugation procedure. The presence of high molecular weight complexes indicated an efficient conjugation procedure. The protocol is simple, and the conjugation steps can be completed in 27 h once the preparatory phase has been carried out; the method is entirely generic and may be applied to labeling of any antibody.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Abuknesha RA,Jeganathan F,Wu J,Baalawy Zdoi
10.1038/nprot.2009.6subject
Has Abstractpub_date
2009-01-01 00:00:00pages
452-60issue
4eissn
1754-2189issn
1750-2799pii
nprot.2009.6journal_volume
4pub_type
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