For sample preparation, 35-day-old Arabidopsis plants were selected for analysis for AsA and glutathione after light treatment. Frozen plant tissues were ground with a mortar and pestle in liquid nitrogen and immediately homogenized with two volumes (w/v) of cold (4 °C) extraction solution (2% trifluoroacetic acid). Then, the homogenates were sonicated on ice and centrifuged at 15,000× g for 20 min at 4 °C. The supernatants were collected, filtered through a 0.45 μm nylon membrane (Millipore, Bedford, MA, USA), and immediately analyzed by LC-UV-MS/MS. All manipulations were carried out in a cold room at 4 °C and protected from light. Each sample was prepared in two replications.
0.45 μm nylon membrane
Manufactured by Merck Group
Sourced in United States
The 0.45 μm nylon membrane is a laboratory filtration product used for the separation and purification of liquid samples. It features a pore size of 0.45 micrometers, which allows the passage of smaller particles and molecules while retaining larger components. The nylon material provides mechanical strength and chemical compatibility for a variety of applications.
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6 protocols using 0.45 μm nylon membrane
1
Ascorbate and Glutathione Analysis in Arabidopsis
2
Quantification of Potato Ascorbic Acid
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A 1 g portion of potatoes thinly pulverized was taken for each preparation technique and was extracted twice with water (10 mL) in an ultrasonic bath for 15 min (Ultracleaner). The pooled extracts were evaporated to dryness under vacuum, and the residues were dissolved in aqueous solution phosphoric acid 0.22 M. The solution was filtered through a 0.45 μm nylon membrane (Millipore) prior to injection into the HPLC-DAD. Separation for qualitative and quantitative analysis of the ascorbic acid was performed according to the method described for anthocyanins and chlorogenic acid [20 (link)]. Detection was carried out at 254 nm according to maximum absorbance wavelength. Results were expressed as mg kg−1 of dry weight. All tests were carried out in triplicates and the standard deviation was calculated for all data.
3
Quantification of Cecal and Fermentation Metabolites
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SCFA (acetate, propionate, butyrate, formate, valerate), branched-chain fatty acids (BCFAs; isobutyrate and isovalerate), and intermediate metabolites (succinate and lactate) concentrations in the cecal and fermentation effluent samples were determined by high-performance liquid chromatography with refractive index detector (HPLC-RI) analysis. Analyses were performed with an Accela Chromatography System and RI detector (Thermo Fisher Scientific Inc., Reinach, Switzerland), equipped with a Security Guard Carbo-H cartridge (4 mm × 3.0 mm) and a Rezex ROA-Organic Acid H + column (300 mm × 7.8 mm). The column was eluted with 10 mM H2SO4 (Fluka) as a mobile phase at a flow rate of 0.4 mL min–1 at 25°C. Cecal samples (500 mg) were mixed with 1 mL of 0.5 mM H2SO4, homogenized, and centrifuged at 4°C at 9,000 × g for 20 min. The supernatant was filtered using 0.45-μm nylon membrane (Millipore AG, Zug, Switzerland) into glass HPLC vials (Infochroma AG, Zug, Switzerland). Reactor effluent supernatant samples were filtered into glass HPLC vials (Infochroma) through a 0.45-μm nylon membrane (Millipore AG) and sealed with crimp caps. SCFAs, BCFAs, and intermediate metabolite concentrations were quantified using external standards (Sigma–Aldrich Chemie).
4
Extracellular Protein Extraction from Bacillus coagulans
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Aliquots from B. coagulans MA-13 strain stored at − 80 °C were grown under standard conditions i.e. in Luria–Bertani liquid medium at 55 °C [5 (link)]. Cells were collected through centrifugation at 3000 × g for 15 min and homogenized by sonication (Sonicator heat system Ultrasonic Inc.) for 10 min, alternating 30 s of pulse-on and 30 s of pulse-off. Clarification of cell extracts was obtained through centrifugation at 40,000 × g for 30 min at 4 °C. For analysis of the extracellular proteins, the supernatant was filtered under vacuum through 0.45 μm nylon membrane (Millipore). The filtrate (secretome) was concentrated 300-fold using an Amicon Ultrafiltration System (Millipore) with a 10 kDa cut-off nitrocellulose membrane (Millipore) at room temperature and a maximum pressure of 75 MPa. Samples were stored at 4 °C for further analysis. At least three independent biological replicates were carried out.
5
Melanin Isolation and Oxidation from P. penguin
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The melanin was isolated from mantle of P. penguin and oxidized as follows (15 (link)). 1 g mantle sample was finely homogenized on ice, mixed with 15 ml PBS (pH 7.4) with 2% (m/V) papain (J&K, China), and incubated at 55°C for 20 h. The precipitate was obtained by centrifuging at 12,000g for 10 min, and then was successively washed with 2 ml mineral ether, ethanol and water. After that, the obtained black precipitate was raw melanin production.
8.6 ml of 1 mol/L K2CO3 and 0.8 ml of 30% H2O2 were used to dissolve and oxidize the raw melanin. The mixture was heated at 100°C for 20 min and cooled down to room temperature. The residual H2O2 was decomposed by 0.4 ml of 10% Na2SO3, and 6 mol/L HCl was then added to adjust pH to 1.0. The mixture was centrifuged at 8,000g for 10 min to get the supernatant, which then was extracted using 70 ml of ether and dried to crystalline residue. Finally, crystalline residues were redissolved in mobile phase or water, and were filtered through 0.45 μm nylon membrane (Millipore, USA) before using.
8.6 ml of 1 mol/L K2CO3 and 0.8 ml of 30% H2O2 were used to dissolve and oxidize the raw melanin. The mixture was heated at 100°C for 20 min and cooled down to room temperature. The residual H2O2 was decomposed by 0.4 ml of 10% Na2SO3, and 6 mol/L HCl was then added to adjust pH to 1.0. The mixture was centrifuged at 8,000g for 10 min to get the supernatant, which then was extracted using 70 ml of ether and dried to crystalline residue. Finally, crystalline residues were redissolved in mobile phase or water, and were filtered through 0.45 μm nylon membrane (Millipore, USA) before using.
6
Quantification of Sugars and FOS in Fermented Extracts
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Fermented extracts were filtered through a 0.45 μm nylon membrane (Millipore) and were used for determination of FOS (1-kestose, 1nystose and 1 F -fructofuranosylnystose), residual sugars, fructose, glucose, and sucrose. Samples were analyzed employing high performance liquid chromatography (HPLC, Perkin Elmer Series 200) with a Prevail Carbohydrate ES Column (5 μm, 250 × 4.6 mm, Grace) at 30 °C. A mixture of acetonitrile/water 70:30 (v/v) was used as mobile phase at a flow rate of 1 mL/min with a pressure of 1700 psi. A refractive index detector (RID) was operated at 35 °C. The response of the RID was recorded and integrated using the TOTALCHROM WS V6.3 software. The quantification of sugars and FOS in samples was determined a through standards curves made with known concentrations of each compound (Nobre et al., 2018) .
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