Product Description of BABEs
Bromoacetamidobenzyl-EDTA (BABE) is a chelate labeling reagent that conjugates with sulfhydryl groups. The iron chelate of BABE (FeBABE) is a unique tool for determining the three-dimensional structure of proteins and the binding structures of protein-protein or protein-DNA complexes. BABE adds EDTA moieties to proteins through their sulfhydryl groups. Once attached to a protein, FeBABE cuts a nearby peptide or DNA chain. The cleavage site is within 12 angstroms of the FeBABE binding site. Iron (II)-chelate cleaves a peptide or DNA chain in the presence of hydrogen peroxide. The cleavage reaction completes quickly: 10 seconds to 20 minutes of incubation is sufficient. The size of the cleaved fragment is analyzed with gel electrophoresis such as SDS-PAGE.
1. Dialyze the protein solution in conjugation buffer (10-20 mM MOPS, 0.2 M NaCl, 2 mM EDTA, 5% glycerol, pH 8.0) at 4ºC overnight.
2. After dialysis, adjust the protein concentration to 15-30 mM.
3. Add 15 ml of 20 mM FeBABE DMSO solution to 1 ml of the protein solution and incubate it at 37ºC for 1 hour. The final concentration of FeBABE is 0.3 mM (10-20X excess to the protein).
4. Dialyze the reaction mixture in protein storage buffer (10-20 mM Tris, 0.1-0.2 M KCl, 10 mM MgCl2, 0.1 mM EDTA, 50% glycerol, pH 7.6) at 4ºC overnight.
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