Hydroxylamine SATA with

Figure 20.13 The thiolation reagent SATA can be used to create sulfhydryl groups on Fab fragments. After deprotection of the acetylated thiol of SATA with hydroxylamine, conjugation with a maleimide-activated enzyme can take place, producing thioether linkages.

Figure 1.63 Deprotection with hydroxylamine of the acetylated thiol of SATA-modified proteins yields a free sulfhydryl group.

Figure 20.6 Available amine groups on an antibody molecule may be modified with the NHS ester end of SATA to produce amide bond derivatives containing terminal protected sulfhydryls. The acetylated thiols may be deprotected by treatment with hydroxylamine at alkaline pH. Reaction of the thiolated antibody with a maleimide-activated enzyme results in thioether crosslinks.

Figure 27.8 SATA may be used to modify a 5 -amine derivative of an oligonucleotide, forming a protected sulf-hydryl. Deprotection with hydroxylamine results in generation of a free thiol.

Figure 57 SATP reacts with amine-containing proteins or other molecules via its NHS ester end to create protected sulfhydryl derivatives in a manner similar to that of SATA. Deprotection can be done with hydroxylamine to free the thiol.

Oligonucleotides containing amine groups introduced by enzymatic or chemical means may be modified with SATA (Chapter 1, Section 4.1) to produce protected sulfhydryl derivatives. The NHS ester end of SATA reacts with a primary amine to form a stable amide bond. After modification, the acetyl protecting group can be removed as needed by treatment with hydroxylamine under mildly alkaline conditions (Fig. 401). The result is terminal sulfhydryl groups that can be used for subsequent labeling with thiol-reactive probes or activated-enzyme derivatives (Kumar and Malhotra, 1992). 

Sulfhydryl groups are introduced using one of two reagents that can be obtained commercially 2-iminothiolane (2-IT) and SATA (Fig. 4). SATA contains a protected sulfhydryl group to confer stability on the molecule. When a free sulfhydryl group is needed, it can be generated by treatment with hydroxylamine. (Fig. 4). 

Therefore, a preliminary study on aliquots of IgG using 2-IT in molar excesses of 10-100 should be performed. When SATA is used the deacetylation of the substituted IgG should be performed with a freshly prepared solution of 1 M hydroxylamine. Sometimes this solution becomes turbid when the pH is brought to 7.5. This is a sign that the reagent is old and should be changed. 

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