Coomassie brilliant blue g 250 protein stain

A. actinomycetemcomitans strain JP2 (Tsai et al., 1984 (link)) was grown in AAGM medium (Fine et al., 1999 (link)). The bacteria were grown on solid AAGM for 48 h at 37 °C in the presence of 10% CO₂. Colonies were inoculated into AAGM broth and were incubated for 24 h. LtxA was purified by ammonium sulfate precipitation from the bacterial culture supernatants as described previously (Diaz et al., 2006 (link)). Purified protein was resolved on a 4–20% SDS-PAGE and visualized by staining with Coomassie Brilliant Blue G-250 protein stain (Bio-Rad Laboratories). The protein concentration was measured with a nanodrop (Thermo Fisher Scientific).

A. actinomycetemcomitans strain JP2 (Tsai et al., 1979 (link)) was grown in AAGM medium (Fine et al., 1999 (link)). After bacteria were streaked on solid media, the plates were incubated at 37°C in the presence of 10% CO₂ for 2 days. Colonies were inoculated into AAGM broth and were grown for 24 hr. LtxA was purified by ammonium sulfate precipitation from the bacterial culture supernatants (Kachlany, Fine, & Figurski, 2002 (link)). The recovered precipitate was suspended in 40 ml of 10-mM KH₂PO₄, pH 6.5, and dialysed overnight. The sample was applied to a HiTRAP^™ SP column (GE Healthcare^™) and then eluted under isocratic conditions with 60% NaCl, 10-mM KH₂PO₄, pH 6.5. Purified protein was resolved on a 4–20% SDS-PAGE and visualized by staining with Coomassie Brilliant Blue G-250 protein stain (Bio-Rad^™). Heat-inactivated LtxA (Δ_HILtxA) was prepared by treatment at 65°C for 30 min. Protein concentrations were determined spectrophotometrically (NanoDrop^™ 2000, Thermo Fisher^™).