Biotin-HPDP

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 6.5-7.5, at a concentration of 2.5 mg/ml. 

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. 

Remove excess biotinylation reagent and reaction by-products by dialysis or gel filtration using a desalting resin. 

Using a similar approach, Clq has been modified with biotin-HPDP and allowed to interact with its specific receptor. Subsequent purification of the Clq receptor was accomplished through cleavage of the disulfide bridge of the biotinylation reagent (Ghebrehiwet et al., 1988). 

Modifications done with biotin-HPDP produce biotinylated compounds with long spacer arms (29.2 A), ensuring good binding efficiency with avidin or streptavidin 

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. 

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Figure 11.9 Biotin-HPDP reacts with sulfhydryl-containing molecules through its pyridyl disulfide group, forming reversible disulfide bonds. The biotin group may be released from modified molecules by reduction with DTT.

Biotin - HPDP (N-[6-(biotinamido)-hexyl]-3 -(2 -pyridyldithio)propionamide)... 

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