EDC plus Sulfo-NHS

Hgure 107 The efficiency of an EDC-mediated reaction may be increased through the formation of a sulfo-NHS ester intermediate. The sulfo-NHS ester survives in aqueous solution longer than the active ester formed from the reaction of EDC alone with a carboxylate. Thus, higher yields of amide bond formation may be realized using this two-stage process. 

In addition to the potential side reactions of EDC as mentioned previously (Chapter 3, Section 1.1), the additional efficiency obtained by the use of a sulfo-NHS intermediate in the process may cause other problems. In some cases, the conjugation actually may be too efficient to result in a soluble or active complex. I rticularly when coupling some peptides to carrier proteins, 1 have found that the use of EDC/sulfo-NHS often causes severe precipitation of the conjugate. Scaling back the amount of EDC/sulfo-NHS added to the reaction may solve this problem. However, eliminating the addition of sulfo-NHS altogether may have to be done in some instances to preserve the solubility of the product. 

The following protocol is a generalized description of how to incorporate sulfo-NHS ester intermediates in EDC conjugation procedures. For specific applications of this technology, the amount of each reagent and unconjugated species may have to be adjusted to obtain an optimal conjugate. 

Dissolve the protein to be modified at a concentration of 1-10 mg/ml in 0.1 M sodium phosphate, pH 7.4. NaCl may be added to this buffer if desired. For the modification of keyhole limpet hemocyanin (KLH Pierce), as described by Staros et al. (1986), include 0.9 M NaCl to maintain the solubility of this high-molecular-weight protein. If lower or higher concentrations of the protein are used, adjust the amounts of the other reactants as necessary to maintain the correct molar ratios. 

Dissolve the molecule to be coupled in the same buffer used in step 1. For small 

Dissolve the molecule to be coupled in the same buffer used in step 1. For small molecules, add them to the reaction in at least a 10-fold molar excess over the amount of protein present. If possible, the molecule may be added directly to the protein solution in the appropriate excess. Alternatively, dissolve the molecule in the buffer at a higher concentration, and then add an aliquot of this stock solution to the protein solution. 

Add the solution prepared in step 2 to the protein solution to obtain at least a 10-fold molar excess of small molecule to protein. 

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