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This is an ELISA-like reaction using BIS-300 kit components to examine the extent of biotinylation of an
AviTag’d protein-of-interest versus a known amount of biotinylated standard. The BRTA procedure
follows a typical ELISA protocol. The standard protein ａnd unknown proteins are adsorbed to the wells
of a 96-well plate at known protein quantities. The biotin associated with the AviTag™ is detected by its
interaction with streptavidin-conjugated alkaline phosphatase. After adsorption, extraneous biotin is
removed by washing the wells, eliminating the need for dialysis steps. NOTE: Some test proteins may
not bind efficiently to the plastic of the 96-well plate.
Reagents needed:
Fully biotinylated MBP-AviTag fusion protein standard (1mg/ml biotinylated MBP-AviTag™; BIS-
300 kit component; Avidity, LLC)
Un-biotinylated MBP-AviTag fusion protein (1mg/ml unbiotinylated MBP-AviTag™; BIS-300 kit
component; Avidity, LLC)
Streptavidin-Alkaline Phosphatase conjugate (Molecular Probes or other)
Solutions:
PBS: 138mM NaCl, 2.6mM KCl in 10mM potassium phosphate (pH 7.4)
PBST: PBS plus 0.05% Tween 20
TBS: 10mM Tris, pH 7.5, 150mM NaCl
Blocking Solution: PBS plus 40μg/mL BSA
Dilution Buffer: PBS plus 0.15μg/mL BSA
Streptavidin-Alkaline Phosphatase conjugate solution: 40μg/mL in PBS
Development Solution for alkaline phosphatase: p-nitrophenyl phosphate in diethanolamine
buffer (Bio-Rad Laboratories)
Stop Buffer: 2M KOH
Equipment /materials:
96-well polystyrene, high binding, flat bottom micro-titer plates (Corning Costar Corp.)
Biokinetics Reader EL312e plate reader (Bio-Tek Instruments) or other.
