ATP Binding Protein Detection Kit – Boehringer Mannheim
Wild or mutant proteins were tested for their ability to bind ATP by using a
covalent ATP analog, the 5P-p-Fluorosulfonylbenzoyl adenosine (FSBA), and a
rabbit anti-FSBA antibody for specific detection.
Three ug of wild or mutant protein in 20 ul is first labelled with 1 mM FSBA in
DMSO for 20 min at 30C. The reaction is stopped by boiling in an equal volume
of sample buffer for 5 min. Proteins are run on SDS polyacrylamide gel. FSBAderivatized proteins are transferred to a nitrocellulose membrane, which is then
treated with 5% milk powder in buffer D (50 mM Tris-HCl (pH 7.4), 150 mM NaCl)
for 1 h. The membrane is incubated with anti-FSBA antibody (Boehringer
Mannheim) diluted 1:500 in buffer D containing 0.1% Tween-20. Secondary
antibody against rabbit is incubated with blot and homemade ECL used to
visualize.