Protein yields of de novo synthesized 14C-leucine labeled Nbs were determined by liquid scintillation counting as described previously (Stech et al., 2012 (link)). In brief, 5 µL aliquots of SN1 or SN2 were precipitated in 3 ml of 10% (v/v) TCA—2% (v/v) casein hydrolysate (Carl Roth GmbH, Karlsruhe, Germany), boiled for 15 min at 80°C and subsequently cooled on ice for at least 30 min. Protein solutions were filtered using a vacuum filtration system (Hoefer, Holliston, United States) and concentrations of 14C-leucine labeled proteins which were retained on the membrane filters (VWR, Darmstadt, Germany) were calculated based on liquid scintillation counting using the Hidex SL600 scintillation counter.
Membrane filters
Manufactured by Avantor
Sourced in Germany
Membrane filters are a type of filtration device used to separate and isolate particles, cells, or molecules from a liquid or gas mixture. They are designed to allow the passage of certain components while retaining others based on their size and physical properties. Membrane filters are commonly used in various scientific and industrial applications, such as water purification, sample preparation, and microbial analysis.
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Lab products found in correlation
5 protocols using membrane filters
1
Measuring Radioactive Protein Yields
2
Phage Isolation and Purification from S. aureus
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Phages were released from S. aureus strains by mitomycin C induction (2.5 µg/ml). Lysates were centrifuged at 8,000 × g for 15 min and the supernatants were passed through 0.45 µm and 0.2 µm membrane filters (VWR International GmbH, Darmstadt). Lytic activity of the phages was tested by spotting 1:10 dilution series of each lysate onto a lawn of MRSA CC398 indicator strains. Phages were isolated by twofold recovery of single plaques. High titres of phages were achieved by harvesting overlay agar with confluent lysis. The agar was resuspended in SM buffer (100 mM NaCl, 8 mM MgSO4 7H2O, 50 mM Tris-HCl, pH 7, 5) and stirred for several hours at room temperature. Thereafter, agar and cell debris were removed by centrifugation and filtration as described above. Phages were concentrated by ultracentrifugation and purified using CsCl step gradients (1,35–1,65 g/cm2). To determine phage titers, the softagar overlay method was applied40 .
3
Synthesis of Carbon Quantum Dots
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Carbon quantum dots were synthesized in a stainless-steel hydrothermal reactor (50 mL volume, 30 bar) (Toption, Shanxi, China) with a Teflon reaction vessel. To the starting material in the amount of 0.5 g (glucose, glucosamine, cellulose and chitosan) 0.5 mL of the hydrochloric acid and 0.1 g of the modifying agent (urea, urotropin) (Sigma-Aldrich, Poznań, Poland) was added. The reactors were placed in oven at 180 °C for 12 h. After the carbonization process suspensions were sonificated using Emmi-20 HC ultrasound bath (EMAG Polska, Juszczyn, Poland) and filtered by using membrane filters (VWR, Gdańsk, Poland) with a 0.22 µm pores diameter. All samples were neutralized by using 5% NaOH solution by using an Elmetron CX-551 pH meter (Elmetron, Zabrze, Poland) and the solutions were dialyzed by using dialysis tubing (MWCO 500–1000 Da) and water (VWR, Gdańsk, Poland) as the purifying agent for 4 days to remove small molecular weight compounds and inorganic ions. Prepared CQDs solutions were diluted for spectroscopic analysis. The reaction parameters and samples composition are given in Table 1 .
4
Thermal Spring Microbiome Characterization
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Four thermal springs differing in their chemical composition and temperature (Additional file 1 : Table S1) were examined in this study, Vřídlo (V, 72.0 °C), Mlýnský (M, 59.3 °C), Sadový starý (S, 46.3 °C), and Štěpánka (P, 18.3 °C). Samples were collected at two time points, in autumn 2018 and spring 2019. A total volume of 25 L of water was collected directly from the constructed pipes into sterile 2 L and 1 L glass bottles (SIMAX, CZ). To control for the asepticity of the collection process, the fallout of possible air contamination was sampled using the same type of 2 L sampling bottle filled with sterile deionized water, which was left open during the whole process of sampling. Collected samples and control bottles were immediately transferred to the laboratory (< 4 h). Cells were filtered onto 0.22 µm membrane filters (VWR, USA), and membrane filters with retained cells from 20 L of water were used for the extraction of metagenomic DNA, whereas filters with retained cells from the 3 L of water were used for the cultivation of microorganisms. The remaining 2 L were used for the enriched cultivation approach.
5
Quantifying Cell-Free Protein Yields
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Total protein yields of cell-free synthesized, 14C-labeled protein were determined by hot trichloroacetic acid (TCA) precipitation and subsequent liquid scintillation counting (Stech et al., 2012 (link)). In short, 3 µl of the fraction analyzed were mixed with 3 ml 10% TCA (Carl Roth GmbH & Co. KG; Karlsruhe, Germany) with 2% casein hydrolysate (Carl Roth GmbH & Co. KG; Karlsruhe, Germany) and incubated in a water bath at 80°C for 15 min followed by 30 min incubation on ice. The TCA solution was then transferred to membrane filters (VWR International GmbH, Darmstadt, Germany) using a vacuum filtration device (Hoefer, Inc., Holliston, USA). The filters were washed with 5% TCA and dried with acetone. The dry filters were transferred into scintillation vials (Sarstedt AG & Co KG) and incubated in 3 ml scintillation cocktail for 1 h. The samples were then measured using the Hidex 600 SL (Hidex; Turku, Finland). Protein yields were calculated based on the counted disintegrations per minute, the specific activity of 14C-leucine in the cell-free reaction, the molecular weight, and the number of leucines in the protein.
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