Amicon ultracel 10k

His₆-RgsD was purified as described previously (57 (link)). Briefly, cells were lysed using M110-L microfluidizer (Microfluidics), and the protein was isolated using a 1-ml HisTrap column (GE Healthcare). Protein was concentrated with Amicon Ultracel-10K (Millipore) to a volume of 2 ml and applied to size exclusion chromatography (SEC) (HiLoad 26/600 Superdex 200 pg; GE Healthcare). After SEC, protein-containing fractions were pooled and concentrated with an Amicon Ultracel-10K (Millipore) according to the experimental requirements.

Staphylococcus aureus core RNAp and σ^A (from pJR28-[6His]rpoD) were prepared exactly as previously described by Reynolds & Wigneshweraraj (16 (link)). Recombinant AgrA was purified as follows: Escherichia coli strain ER2566 containing pSN-agrA was grown at 37°C. At OD_{600 nm} ∼0.6, the cells were temperature shifted to 16°C for 30 min, and the expression of AgrA was induced with 0.25 mM IPTG. The cells were harvested after 17 h at 16°C. AgrA was purified using the IMPACT^TM kit (New England Biolabs) according to the manufacturer's instructions. Briefly, the cells were lysed in a buffer containing 20 mM Tris-HCl (pH9), 1 M NaCl and 1 mM EDTA (column buffer) and centrifuged to remove cellular debris. The supernatant was then loaded on a 10 ml gravity flow column (Bio-Rad) packed with 2 ml Chitin Resin (New England Biolabs). The column was washed with 20 bed volumes of column buffer and the protein was cleaved from the intein tag after incubation for 16 h at 4°C in three bed volumes of cleavage buffer [column buffer + 200 mM DTT]. The protein was concentrated using Amicon Ultracel-10K (Millipore) and dialysed in a storage buffer [10 mM Tris-HCl (pH8), 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT and 20% (vol/vol) glycerol].