Journal: The Journal of Biological Chemistry

Article Title: Identification, Characterization, and Structure Analysis of the Cyclic di-AMP-binding P II -like Signal Transduction Protein DarA *

doi: 10.1074/jbc.M114.619619

Figure Lengend Snippet: Identification of the c-di-AMP receptor DarA in B. subtilis. A, a pulldown assay was performed with B. subtilis crude extract to identify c-di-AMP-binding proteins. After incubation of Strep-Tactin-covered beads with 2′-AHC-biotinylated c-di-AMP and a B. subtilis crude extract, the samples were washed to eliminate nonspecifically bound proteins. Elution was performed with 5 mm biotin. Samples were analyzed by SDS-PAGE with subsequent silver staining. As the negative control, the crude extract was incubated with Strep-Tactin-covered beads alone (−). B, DarA was overexpressed in E. coli and subsequently incubated with 2′-AHC-biotinylated c-di-AMP. After incubation, the sample was purified using a Strep-Tactin column, and bound protein was eluted with 2.5 mm d-desthiobiotin. As a negative control, the overexpressed protein was purified without addition of 2′-AHC-biotinylated c-di-AMP (−). The last wash fraction (W7) and elution fractions (E1–E3) were analyzed by SDS-PAGE with subsequent silver staining.

Article Snippet: To isolate c-di-AMP-binding proteins from B. subtilis , biotinylated c-di-AMP (2′-[biotin]-AHC-c-di-AMP; BIOLOG, Bremen, Germany) was coupled to Strep-Tactin-covered MagStrep “type 2HC” beads (IBA, Göttingen, Germany).

Techniques: Binding Assay, Incubation, SDS Page, Silver Staining, Negative Control, Purification