Biotin-BMCC

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. 

Biotin-BMCC is insoluble in water and must be dissolved in an organic solvent prior to addition to an aqueous reaction mixture. Preparing a concentrated stock solution in DMF or DMSO allows transfer of a small aliquot to a buffer reaction. The upper limit of biotin-BMCC solubility in DMSO is approximately 33 mM or 17 mg/ml. In DMF, it is only soluble to a level of about 7 mM (4 mg/ml). Upon addition of an organic solution of the reagent to an aqueous environment (do not exceed 10 percent organic solvent in the aqueous medium to prevent protein precipitation), biotin-BMCC may form a micro-emulsion. This is normal and during the course of the reaction, the remainder of the compound will be driven into solution as it couples or hydrolyzes. 

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. 

The required sulfhydryl groups for biotin-BMCC modification may be indigenous 

Dissolve the protein to be biotinylated (containing one or more free sulfhydryls) in 0.1 M sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2, at a concentration of 2.5 mg/ml. 

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Figure 11.8 Biotin-BMCC provides sulfhydryl reactivity through its terminal maleimide group. The reaction creates a stable thioether linkage.

Dissolve biotin-BMCC (Thermo Fisher) in DMSO at a concentration of 5 mg/ml. 

Add 100 pi of the biotin-BMCC solution to each ml of the protein solution. Mix well. 

Reaction of Biotin-BMCC with Sulfhydryl-Modified DNA... 

Figure 27.12 Biotin-BMCC can be used to modify a reduced, cystamine derivative of DNA, forming a thioether linkage.

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