antibody biotinylation (fwd)

Hao Xiao haoxiao at cc.UManitoba.CA
Mon Oct 31 22:54:44 EST 1994


Mr. M. Kuiper writes:
> From BIOSCI-REQUEST at net.bio.net Mon Oct 31 10:51:01 1994
> To: immunology at net.bio.net
> From: mkuiper at hgmp.mrc.ac.uk (Mr. M. Kuiper)
> Subject: antibody biotinylation
> Date: 31 Oct 1994 16:14:55 -0000
> Sender: lpddist at mserv1.dl.ac.uk
> Message-ID: <39355v$86v at mserv1.dl.ac.uk>
> Original-To: immuno at dl.ac.uk
> 
> Dear immuno-netters,
> 
> Can anybody out there tell me whether it is possible to
> biotinylate antibodies yourself. If anyone could suggest
> an efficient protocol I would be most grateful. I want
> to use the biotinylated antibodies in ELISAs.

very easy, follow my simple procedure :

1) adjust your Ab (10 mg in 1 ml) solution to a pH of 8.5 with protasium
phosphate (dibase).

2) disolve 1 mg of biotin-N-hydroxylsuccinimide ester with 100 ul of DMSO
and add dropwise to your Ab solution at 25C for 2 hr.

3) pass the mixture through a G10 sephadex column, discard the void volume
then begin to collect 120% of your original adding volume.

4) you will get 10-20 molecules of biotin in one molecule of Ab.

5) if this procedure does not work, blame me.


Hao Xiao

haoxiao at cc.umanitoba.ca 
Ph.D student in Pharmacology and Toxicology
Univeresity of Manitoba

> 
> Thanks for your help,
> *******************************************************************
> *                                                                 *
> *	Marcel Kuiper                                             *
> *	King's College School of Medicine & Dentistry             *
> *	Department of Molecular Medicine                          *
> *	Rayne Institute, 2nd floor                                *
> *	123 Coldharbour Lane                                      *
> *	London SE5 9NU, UK                                        *
> *	Fax:	44-71-733 3877                                    *
> *	Email:	mkuiper at hgmp.mrc.ac.uk                            *
> *		rchz103 at bay.cc.kcl.ac.uk                          *
> *                                                                 *
> * 'The first myth of management is that it exists'. Murphy's Law. *
> *                                                                 *
> *******************************************************************
> 
> 




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