In NHS ester reactions

Prepare the protein to be modified in a non-amine-containing buffer at a slightly basic pH (i.e., avoid Tris or imidazole). The use of 0.1 M sodium phosphate, 0.15M NaCl, pH 7.2 works well for NHS ester reactions. The concentration of the protein in the reaction buffer may vary from pg/ml to mg/ml, but highly dilute solutions will result in less efficient modification yields. A protein concentration from 1 to 10 mg/ml works well in this reaction. 

Fluorophenyl esters react with amine-containing molecules at slightly alkaline pH values to give the same amide bond linkages as NHS esters (Reaction 15). However, in most cases, the fluorophenyl ester compound will display better stability toward hydrolysis in aqueous solution. It has been reported that a TFP ester has over twice the half-life in basic pH buffers (pH 8) than a corresponding NHS ester on the same compound (Molecular Probes). 

Dissolve the protein to be modified in 50mM sodium borate, pH 8.5, at a concentration of lOmg/ml. Other buffers may be used for an NHS ester reaction, including 0.1M sodium phosphate, pH 7.5 (Chapter 2, Section 1.4). 

Figure 16 Conjugation of an amine and a carboxylic acid via the (V-hydroxysuccinimide (NHS)-activated ester method. NHS esters may be isolated and characterized and are stable to long term storage as the powder. Alternatively, the NHS esters may be used immediately upon formation without isolation. Details of the reaction are given in Table 4...

Dissolve the protein to be thiolated at a concentration of l-5mg/ml in 50 mM sodium phosphate, pH 7.5, containing 1-10 mM EDTA. Other non-amine containing buffers such as borate, HEPES, and bicarbonate also may be used as the reaction medium. The effective pH for the NHS ester modification reaction is in the range of 7.0-9.0, but environments closer to neutrality will limit the hydrolysis of the ester. 

Figure 1.113 The NHS ester-suberate derivative of lactose can be used to add lactose groups to amine-containing molecules. The reaction results in the formation of amide bonds containing terminal lactose groups.

Dissolve mono(lactosylamido) mono(succinimidyl)suberate in dry DMF to prepare a concentrated solution from which an aliquot may be taken and added to a final aqueous reaction medium. The compound is extremely soluble in DMF, and solutions of 100 mg/ ml may be prepared. The use of dry solvent is essential to prevent hydrolysis of the NHS ester. However, make only enough of this stock solution so that a small amount added to the protein reaction will provide the appropriate molar excess desired for the modification reaction. 

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