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J Biomol Tech. 2007 February; 18(1): 46–47.
PMCID: PMC2292025

P133-S An Improved Protocol of Coupling Synthetic Peptides to KLH for Antibody Production Using DMF


Keyhole limpet hemocyanin (KLH) conjugated peptides are routinely used as antigens to generate antibody. In the traditional method, peptides are dissolved in phosphate-buffer saline (PBS) and then mixed with 3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) conjugated KLH to make KLH conjugated peptides. If the peptide is not soluble in PBS, it can be dissolved in 6 M guanidine-HCl/0.01 M phosphate buffer, pH 7.0. However, there are peptides which are insoluble in both buffers. We present here a procedure of making the KLH conjugated peptides using dimethylformamide (DMF) instead of PBS or 6 M guanidine-HCl to increase solubilities of peptides. Additionally, the last desalt step in the traditional method is eliminated. Briefly, 5 mg of peptide is dissolved in 100 μL of DMF. This solution is added to 1 ml of purified KLH-MBS slowly. The pH of the solution is adjusted to ~7.0 immediately by adding either 2 N NaOH or 0.5 N HCl. The solution is then stirred or rotated for 3 h or overnight at 4°C. Many peptides which do not dissolve in 6 M guanidine-HCl will be soluble in DMF. Some peptide solutions may give cloudy or gelly appearance, nevertheless, peptides do react with KLH to some extend and solutions will become clear. The reaction can be kept longer for such peptides. Also, some peptide-KLHs do form precipitates which can be left in the final solution. Finally, 2 ml of 0.1 M ammonium bicarbonate is added to the peptide/KLH solution, and the solution is lyophilized. Compare to the traditional method, our method is faster and peptides with solubility problems in PBS or 6 M guanidine-HCl can be used.

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