1. It is often worth adding a cysteine residue at the C-terminus of the peptide to give the option of coupling via MBS as well as via glut-araldehyde.

2. During glutaraldehyde conjugation it is vital to exclude any buffers containing amino, imino (e.g., Tris-HCl), ammonium or azide moieties as these will inhibit the cross-linking reaction. If the peptide or carrier is insoluble in coupling buffer, sodium dodecyl sulphate may be added to 0.1% without affecting the conjugation. Occasionally, a peptide-carrier conjugate becomes less soluble as the conjugation reaction proceeds. This does not appear to affect the efficacy of the final product and is usually, therefore, no cause for concern.

3. If, during MBS coupling, the DMF concentration exceeds 30% the KLH will come out of solution. KLH concentrations in excess of 20 mg/mL will also lead to insolubility. Use the Sulfo-MBS derivative if possible.

4. If many conjugation reactions are required, the MBS activation of KLH can be scaled up and performed batchwise. Alternatively, if only a few conjugates are needed, Maleimide-activated KLH is commercially available (Calbiochem or Pierce). This leaves only the final addition of peptide to be performed.

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