Amines with SATP

Dissolve the protein or peptide to be thiolated at a concentration of lOmg/ml in 50 mM sodium phosphate, pH 7.5, containing 1 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. Conditions closer to neutral pH will limit the degree of NHS ester hydrolysis during the reaction. 

Dissolve the SATP reagent (Molecular Probes) in DMF at a concentration of 65 mM (16mg/ml). Note DMF should be handled in a fume hood. 

Add 10 pi of the SATP solution to each ml of protein or peptide solution. 

Deprotect the acetylated —SH groups as needed by adding 100 pi of 0.5 M hydroxy-lamine hydrochloride in 50mM sodium phosphate, 25 mM EDTA, pH 7.5, to each ml of the SATP-modified protein solution. 

Purify the sulfhydryl-modified protein by dialysis against 50mM sodium phosphate, 1 mM EDTA, pH 7.5, or by gel filtration on a Sephadex G-25 column using the same buffer. Again, if a peptide of low molecular weight is being modified, use careful gel filtration for purification. 

Add 10 xl of the SATP solution to each milliliter of protein or peptide solution. Mix and react for 8 h (or overnight) at room temperature. 

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Figure 1.65 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 hydroxy-lamine to free the thiol.

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