SATA-Modified Antibodies

Deprotect the acetylated sulfhydryl groups on the SATA-modified antibody according to the following protocol ... 

Add 100 xl of the hydroxylamine stock solution to each milliliter of the SATA-modified antibody. Final concentration of hydroxylamine in the antibody solution is 50 mM. 

Dissolve the antibody to be modified in 0.1 M sodium phosphate, 0.15M NaCl, pFl 7.2, at a concentration of 1-5 mg/ml. Note Phosphate buffers at various pFl values between 7.0 and 7.6 have been used successfully with this protocol. Other mildly alkaline buffers may be substituted for phosphate in this reaction, providing they don t contain extraneous amines (e.g., Tris) or promote hydrolysis of SATA s NHS ester (e.g., imidazole). 

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.

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