Tris or Ethanolamine plus EDC

Dissolve the macromolecule containing carboxylate groups to be blocked at a concentration of l-10mg/ml in 0.1 M MES, pH 4.7, containing 0.1 M Tris or ethanolamine. Other conditions may be used to perform this reaction. See Chapter 3, Section 1 for further details. 

This section is designed to provide a general overview of activation and coupling chemistry. Some of the reagents discussed in this chapter are not themselves crosslinking or modification compounds, but may be used to form active intermediates with another functional group. These active intermediates subsequently can be coupled to a second molecule that possesses the correct chemical constituents, which allows bond formation to occur. 

Ultimately, this section is meant to function as a ready-reference database for learning or review of bioconjugate chemistry. In this regard, a reaction can be quickly found, a short discussion of its properties and use read, and a visual representation of the chemistry of bond formation illustrated. What this section is not meant to be is an exhaustive discussion on the theory or mechanism behind each reaction, nor a review of every application in which each chemical reaction has been used. For particular applications where the chemistries are employed, cross-references are given to other sections in this book or to outside literature sources. 

Isothiocyanate compounds react best at alkaline pH values where the target amine groups are mainly unprotonated. Many reactions are done on 0.1M sodium carbonate buffer at pH 9. Reaction times vary from 4 to 24 h at 4°C. Since the isothiocyanate group is relatively unstable in aqueous conditions, reagents containing this function should be stored desiccated at refrigerator or freezer temperatures. 

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