HABA assay

Determination of the degree of biotinylation can be done using the HABA assay (Chapter 23, Section 7). 

Proteins biotinylated with this reagent will have a characteristic absorbance band at 354 nm, which can be used to determine accurately the number of biotin groups per molecule. No other biotinylation compound has such built-in quantification capability. This feature eliminates the need to consume conjugate by doing a HABA assay to test for the level of biotin incorporation (Chapter 23, Section 7). 

A brief discussion of the limitations of the HABA assay is found in Note 8. 

New rhenium(I) biotin complexes containing the extended planar diimine ligands dppz and dppn (17) have been prepared to explore bifunctional biological probes [44], These complexes bind to double-stranded calf thymus DNA by intercalation, as revealed by absorption and emission titrations. HABA assays show that all the complexes bind to avidin with a stoichiometry of 4 1 ([Re] [avidin]). Similar to other rhenium(I) diimine biotin complexes described above, the emission intensities... 

Four luminescent cyclometallated iridium(III) diimine complexes [Ir(ppy-spacer-biotin)2(NAN)]+ (NAN = Me4-phen, Ph2-phen) (38), each containing two biotin units, have been synthesised and characterised by Lo and co-workers [79], Photoexcitation of these iridium(III) diimine bis(biotin) complexes in fluid solutions at 298 K and in alcohol glass at 77 K results in intense and long-lived 3MLCT (djr(Ir) — ti (NaN))/3IL (ir —> ji ) (Me4-phen) emission. HABA assays and emission titrations indicate that the two biotin moieties of each complex are functional. RET-based emission-quenching experiments, microscopy studies using avidin-conjugated microspheres, and HPLC analysis all reveal that the complexes with a... 

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