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.