# Role of Hibernation Promoting Factor
## 4 Materials and Methods
### Strains
The purified protein eluates were monitored by the Bicinchoninic Acid (BCA) Assay (Pierce Biotechnology, Waltham, MA, USA), absorbance at 562 nm on the Nanodrop1000 (Thermo Fisher Scientific, Rockford, IL, USA), and SDS-PAGE using Coomassieie blue staining. Additionally, proteins were assayed with immunoblots using an antibody to the 6x-poly-histidine tag (Thermo Fisher Scientific, Rockford, IL, USA) with chemiluminescent detection, as described previously [52]. **Antibody Generation and Purification. ** Poly-clonal antisera to L5 and S13 proteins were raised in rabbits by Lampire Biological Laboratories (Pipersville, PA, USA). The IgG fraction of the antisera was purified using NAbTM Protein A Plus Spin Columns (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer's instructions. Polyclonal antisera to HPF was purified as described previously [22]. The efficacies of the purified antibodies were tested using immunoblots against the purified protein, against _P. aeruginosa_ PAO1 protein extracts, and against extracts of _P. aeruginosa_ containing fusions of the ribosomal proteins to the yellow fluorescent protein. **Ribosome purification and analysis. ** Ribosomes were partially purified from _P. aeruginosa_ PAO1 and _P. aeruginosa_ Alnpf_ cells before and after starvation in phosphate buffered saline (PBS) pH 7.0. For these experiments, 1 mL of overnight culture was added to 100 mL Trypic Soy Broth (TSB). The cultures were incubated at 37 \({}^{\circ}\)C with 200 rpm shaking until the OD\({}_{600}\) reached 6.0 on a CE2041 Spectrophotometer (Cecil Instrumentation Services Ltd., Milton, Cambridge, UK). Cells were washed twice in PBS, resuspended in 10 mL PBS, and then added to 100 mL of PBS to give a final OD\({}_{600}\) of 1.0, corresponding to approximately \(2.4\times 10^{9}\) CFU/mL. The 110 mL samples were collected immediately or following 6 days of incubation with shaking at 200 rpm at 37 \({}^{\circ}\)C. For sample collection, the cells in PBS were centrifuged at 8000\(\times\)\(g\) for 10 min at 4 \({}^{\circ}\)C, washed once in PBS, and transferred to 1.5 mL microcentrifuge tubes. Cells were further concentrated by centrifugation at 9500\(\times\)\(g\) for 5 min at 4 \({}^{\circ}\)C. The supernatant was removed and the cell pellets were frozen at -80 \({}^{\circ}\)C. The cells were resuspended in 50 \(\mu\)L ice-cold Buffer A [53], brought to 1 mL with a final concentration of 0.7 mM lysozyme, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.5\(\times\)CellyticTM B (Sigma-aldrich, Burlington, MA, USA), 3 mM Magnesium, and diethyl pyrocarbonate (DEPC) treated water. Cells were lysed by eight freeze-thaw cycles consisting of 10 min at \(-\)80 \({}^{\circ}\)C followed by 2 min in a 37 \({}^{\circ}\)C water bath. Ten units of RNase-Free DNAse (New England Biolabs, Ipswich, MA, USA) was added to each cell lysate and five additional freeze-thaw cycles were performed to ensure complete lysis. The cell lysates were spun at 20,000\(\times\)\(g\) for 45 min at 4 \({}^{\circ}\)C to remove cellular debris. The supernatants were collected and ribosomes were separated using Buffers A, B, and C from [53] according to the previously published crude preparation protocol in [54]. Briefly, the supernatants were adjusted to a final volume of 6 mL with Buffer A and slowly added to RNase-free ultracentrifuge tubes (Beckman Coulter, Indianapolis, IN, USA) containing 6 mL Buffer B. The fractions were centrifuged at 111,132\(\times\)\(g\) for 15 h at 4 \({}^{\circ}\)C using the SW41 Ti rotor in an OptimaTM L-100 XP Ultracentrifuge (Beckman Coulter, Indianapolis, IN, USA). The supernatants were carefully removed, and the ribosome pellets were resuspended in 200 \(\mu\)L Buffer C.  
To assay for rRNA in ribosome preparations, 2 \(\mu\)L aliquots of purified ribosomes were run on agarose gels containing bleach as described previously [55].