Re: peptide conjugation
Dan Crimmins (crimmins@pathbox.wustl.edu)
Wed, 20 Jan 1999 08:39:42 -0600
At 11:15 AM 1/20/99 +1100, Michael Jobling wrote:
>Hi everybody.
>We are trying to conjugate KLH to a peptide (via a free cysteine). The
>problem is that our peptide has two cysteine residues which form a
>disulphide bond quite readily. After reduction by DTT, the disulphide
>reforms during dialysis to remove the DTT from the reaction mixture. Apart
>from running a column or 'quicker' method of removing the DTT, so the
>disulphide doesn't have a chance to reform, does anybody have a suggestion
>as to how we might be able to maintain a free S-H to add the KLH to.
>Thanks in advance
>
>
>Michael Jobling
>Dept. of Pathology
>Uni. of Melbourne
>m.jobling@pathology.unimelb.edu.au
>
>
>
>
Michael,
If you are using NEM-KLH for instance,then you clearly need free thiol
groups on the peptide as you stated. To maintain the thiol groups in the
reduced and therefore reactive from you can use TCEP which should not react
with the target site(s) on KLH.
Regards,
Dan L. Crimmins
Washington University School of Medicine
Dept. Pathology/Division of Laboratory Medicine
660 S. Euclid Ave., Box 8118
St. Louis, MO 63110
Phone: 314-454-8514; Fax: 314-454-5208
e-mail: crimmins@labmed.wustl.edu