Sulfhydryls biotinylation

Sulfhydryl groups also can be added to 5 -phosphate end of DNA probes (Chapter 27, Section 2.2). Biotinylation at these sites avoids disruption of base pairing with complementary DNA targets, since the point of modification is restricted to a single end position on the oligonucleotide. 

The reagent is similar to another maleimide-containing biotinylation reagent, 3-(N-maleimi-dopropionyl) biocytin, a compound used to detect sulfhydryl-containing molecules on nitrocellulose blots after SDS-electrophoresis separation (Bayer et al., 1987). Biotin-BMCC should be useful in similar detection procedures. 

The following protocol is a suggested method for modifying sulfhydryl-containing proteins with biotin-BMCC. Some optimization of biotinylation levels may have to be done for particular applications. 

A suggested protocol for the use of biotin-HPDP in the modification of sulfhydryl-containing proteins follows. Similar procedures may be used when biotinylating other molecules. 

Figure 11.10 This biotinylation reagent reacts with sulfhydryl groups through its iodoacetamide end to form thioether bonds.

Dissolve a sulfhydryl-containing protein or other thiol-molecule in a thiol-free buffer within a pH range of 6.5-7.5. The use of 20 mM sodium phosphate, 150 mM NaCl, pH 7.2, works well for this reaction. The concentration of protein should be in the range of l-10mg/ml. Lower concentrations of protein may result in the need to increase the molar excess of biotinylation reagent to obtain an acceptable level of modification. If a... 

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