Cellulose nitrate membrane filter

Each sample (1000 mL) was filtered through cellulose nitrate membrane filters (47 mm Ø and 0.45-mm pore size; Millipore, Milan, Italy). The membrane was then immersed in Buffered Peptone Water (BPW) (Biolife Italiana srl, Milan, Italy) and incubated for 18–24 h at 36 ± 1 °C. Subsequently, a 0.1-mL aliquot of culture was transferred to 10 mL of Rappaport Vassiliadis broth (Microbiol & C. s.n.c, Uta, Italy) and incubated for 24 + 24 h at 41.5 °C. The broth was then streaked after 24 and 48 h on xylose lysine deoxycholate agar plates (Biolife Italiana srl) and Hektoen Enteric Agar (Merck, Darmstadt, Germany), respectively. After 24 h at 36 ± 1 °C, colonies with typical morphology were streaked on Tryptic Soy Agar plates (Biolife Italiana srl), incubated at 36 ± 1 °C for 24 h and subjected to biochemical confirmation tests (API 20E, Biomèrieux, Marcy l’Etoile, France). Finally, typing through specific serological tests [56 ] was performed.

cGMP binding assay was performed in a total volume of 100 µl of 20 mM Tris-HCl (pH 7.5) buffer containing 100 mM NaCl, 2 mM 2-mercaptoethanol, 1 mM EDTA, 10 pmol of PDE6β1-313, [H³]cGMP (∼100,000 cpm) and varying concentration of unlabeled cGMP. The binding reactions were incubated for 2 hrs at 4°C and then applied onto pre-wet 0.45 µm cellulose nitrate membrane filters (Millipore). The filters were washed twice with1 ml of cold PBS buffer (pH 7.5) containing 1 mM EDTA, and counted by liquid scintillation counting. The data were fit to equation for binding with ligand depletion.[22] (link)

Reassociated 70S ribosomes were prepared from E. coli K12 cells as described previously [22 (link)]. 70S ribosomes were incubated in buffer A (100 mM Tris-HCl pH 7.2, 100 mM ammonium acetate, 10 mM magnesium acetate, 6 mM β-mercaptoethanol) with 10 μΜ [¹⁴C]-chloramphenicol (150 dpm/pmol) at a final concentration of 0.20 μM [23 (link)]. After incubation for 10 min at 37 °C, the mixture was diluted with 3 mL of cold buffer A and filtered through a 25-mm diameter cellulose nitrate membrane filter (Millipore 0.45 μm pore size). The filter was washed three times with 3 mL of cold buffer A and the radioactivity which remained bound on the filter was measured. The binding of [¹⁴C]-chloramphenicol was studied in competition with CAM or PA–CAM conjugates by keeping the concentration of [¹⁴C]-CAM constant (10 μM) and increasing the concentration of non-radioactive conjugates [23 (link)].