Purified 70S WT or Cfr-modified ribosomes were diluted to 1 pmol μl−1 in buffer containing 50mM Hepes-KOH (pH 7.5), 150mM KOAc, 20mM Mg(OAc)2, 7 mM β-mercaptoethanol and 20Uml−1 SuperASE-In. Diluted ribosomes (50 μl) were then incubated at 4 °C for 1.5 h after the addition of 3 μl of either 1mM LZD (Med Chem Express) or 1mM RZD (Med Chem Express), yielding a final antibiotic concentration of 60 μM (60× molar excess). Samples were then filtered for 5 min at 14,000g at 4 °C using a 0.22-μm low-binding Durapore PVDF filter (Millipore).
Durapore pvdf filter
Manufactured by Merck Group
Sourced in United States
Durapore PVDF filter is a laboratory filtration product manufactured by Merck Group. It is designed for general filtration applications in research and industrial settings. The filter is made of polyvinylidene fluoride (PVDF) material and provides reliable performance for a variety of liquid filtration needs. The key function of the Durapore PVDF filter is to separate and retain particles, microorganisms, or other suspended solids from liquid samples.
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Lab products found in correlation
Zorbax sb c18 column, by Agilent Technologies (1 mentions)
Agilent 1200 hplc system, by Agilent Technologies (1 mentions)
Cs110 4, by Labogene (1 mentions)
Genesys 10s uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Tecnai g20 electron microscope, by Thermo Fisher Scientific (1 mentions)
Axis his spectrometer, by Shimadzu (1 mentions)
Dimension 3100, by Veeco (1 mentions)
4 protocols using durapore pvdf filter
1
Ribosome Antibiotic Binding Assay
2
Quantifying Resveratrol in Transgenic Vitis
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The accumulation of resveratrol and its stilbenoid derivates were analyzed in triplicate in PM-inoculated and mock-inoculated leaf samples (0.5 g each, 7 dpi) harvested from 4-week old VqSTS21 transgenic lines and untransformed controls. The resulting samples were ground to a fine powder in liquid nitrogen using a mortar and pestle. Ground leaves were extracted with 5 ml 80% methanol, and supernatant fractions were collected following centrifugation at 4500 g for 5 min. The resulting extracts were evaporated using a vacuum rotary evaporator (CS110-4, LaboGene, Denmark), and were immediately re-dissolved in 0.2 ml of pure methanol. These extracts (30 μl) were then filtered through a 0.45 μm sterile Durapore® PVDF filter (Millipore, USA). Samples were run on an Agilent 1200 HPLC system (Agilent, Waldbronn, Germany) with an Agilent ZORBAX SB-C18 column (5 μm, 4.6 × 250 mm), H2O-methanol as eluent (H2O:methanol [60:40], flow rate 0.8 ml/min), and a wavelength of 306 nm for detection. The column temperature was maintained at room temperature. Stilbenoids in transgenic lines were identified by comparing the retention time with those of standards.
3
Outer Membrane Vesicle Isolation
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The E. coli F4 and F18 serotypes used in this study were obtained from CECT (Valencia, Spain) and Agropecuaria Obanos (Navarra, Spain), respectively. Strains were cultured in Tryptone–Soya–Broth for 18 h at 37 °C with agitation. OMVs were obtained following a method adapted from Camacho et al. [22 (link)]. Bacteria were grown in 500 mL of TSB under shaking overnight to early stationary phase (37 °C, 125 rpm). Then, bacteria were inactivated during 6 h with a solution of binary ethylenimine and formaldehyde (6 mM BEI—0.06% FA, 6 h, 37 °C). Cells were discarded by centrifugation (10,000× g, 10 min) and the supernatant filtered through a 0.45 µm Durapore PVDF filter (Millipore) and purified by tangential filtration using a 300 kDa concentration unit (Millipore). The retenate was frozen and subsequently lyophilized.
4
Exfoliation and Characterization of BEGO
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The exfoliation mixture was first centrifuged at 2500 rpm to remove large graphite particles and possible planktonic microorganisms, and then the supernatant was filtered through a 0.1 μm Durapore PVDF filter (Millipore Co.,). The remaining solid materials on the filter were washed with the following sequence: 18 MΩ water, 80% ethanol, 18 MΩ water, 1 N HCl, and 18 MΩ water again. The resulting materials called BEGO were re-suspended in 18 MΩ water for characterization. UV-vis absorption spectra were recorded using a Thermo GENESYS 10S UV-vis spectrophotometer. Fourier transform infrared spectroscopy (FT-IR) was performed by using a Thermo-Nicolet FT-IR Avatar 370 spectrometer. XPS analysis was carried out by a Kratos Axis His spectrometer. Raman spectra were obtained by Raman Microscope with wavelength of 532 nm and a 100 × objective. Atomic force microscopy (AFM) images were generated on a VEECO Dimension 3100. Transition electron microscopy (TEM) and electron diffraction images were obtained using a FEI Tecnai G20 electron microscope. Samples for AFM and TEM imaging were prepared by drop-casting the dispersion onto freshly cleaved mica substrates and lacey carbon TEM grid, respectively, which were then air dried under ambient lab conditions.
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