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 μl 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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Erin L. Benanti1 and Peter T. Chivers, Helicobacter pylori NikR Protein Exhibits Distinct Conformations When Bound to Different Promoters, THE JOURNAL OF BIOLOGICAL CHEMISTRY, 2011, 286, 15728.
Chih-Chien Wu, Yu-Chun Lin, and Hung-Ta Chen, The TFIIF-Like Rpc37/53 Dimer Lies at the Center of a Protein Network To Connect TFIIIC, Bdp1, and the RNA Polymerase III Active Center, MOLECULAR AND CELLULAR BIOLOGY, 2011, 31, 2715.
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