Three independent cultures of control and Tfe2-expressing S. cerevisiae cells were incubated for 6 h at 30°C in galactose induction media, washed twice with ice-cold water, normalized to 5 OD600 per 1 ml of water and boiled for 15 min. After centrifugation, the supernatant was clarified using a 0.2 µm syringe filter (Sartorius). The resulting extract was acidified by addition of TFA in MeOH (10%, v/v), dried and resuspended in buffer (40% 1M NaOAc, 40% MeOH, 20% triethylamine, TEA), before being dried again. The amino acids were then derivatized using Phenylisothiocyanate (PITC) by resuspension of the dried material in 10% PITC, 70% MeOH, 10% H2O, 10% TEA, followed by 2-fold dilution in MeOH. After 15 min incubation at room temperature, suspensions were dried, washed once with MeOH and suspended in elution buffer, composed of 95% (150 mM NaOAc, 0.5 ml/l TEA, pH 6.4) and 5% acetonitrile. PTC amino acid derivatives were analysed by HPLC using a Nova-Pak® C18 column (Waters), the Clarity Lite software and two amino acid standard sets (AA-S-18 and A6282, Sigma). The experiment presented has been reproduced on two independent occasions.
0.2 m syringe filter
Manufactured by Sartorius
Sourced in Germany
The 0.2 μm syringe filter is a laboratory filtration device designed to remove particles and microorganisms from liquid samples. It features a 0.2 μm pore size membrane that allows the passage of the liquid while retaining particulates and microbes. The filter is intended for use in a variety of laboratory applications that require sterile filtration.
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5 protocols using 0.2 m syringe filter
1
Quantitative Amino Acid Analysis in Yeast
2
Synthesis and Characterization of SPIONs
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SPIONs were synthesized as described previously using an adjusted protocol by Elbialy et al. (23 (link), 32 (link)) The nanoparticles were sterilized by filtration through a 0.2 µm syringe filter (Sartorius, Goettingen, Germany). The hydrodynamic size, iron concentration, magnetic susceptibility, and zeta potential of the SPIONs were then characterized as described by Mühlberger et al. and Boosz et al. (21 (link), 23 (link)) The iron content was investigated at a dilution of 1:25 in dH2O, dissolved in 65% nitric acid, using atomic emission spectroscopy with an Agilent 4200 MP-AES (Agilent Technologies, Santa Clara, CA, USA) with an iron solution of 1000 mg Fe/L as an external standard (Bernd Kraft, Duisburg, Germany). Measurements were performed in triplicates at a wavelength of 371.993 nm, which were then averaged. SPIONs were diluted with sterile dH2O to the intended concentration for all experiments.
3
Antimicrobial Peptides against M. tuberculosis
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Both L- and D-amino acids of hLF 1-11 and L-enantiomer of PG-1, AK 15-6, and AM-mel are purchased from BioBasic Inc. (Amherst, NewYork, USA) and Synpeptide Co., Ltd. (Shanghai, China). The antimycobacterial activity of antimicrobial peptides, PG-1, AK 15-6, and AM-mel, have previously been elucidated. These peptides are used as positive control in this study. The general characteristics and the previously reported MIC of each peptide against M. tuberculosis are summarized in Table 6 . Each peptide with a purity of over 95% is dissolved in sterile phosphate buffer saline (PBS), pH 7.0, filtered using 0.2 µm syringe filter (Sartorius, Germany), aliquoted and kept at −20 °C.
4
Glucose and Nitrate Analysis by HPLC and IC
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The supernatant from the culture was filtered using a 0.2 µm syringe filter (Sartorius Stedim Biotech, Germany). A high-pressure liquid chromatograph (HPLC, Waters, USA) equipped with a refractive index detector (RI) was used to analyze the glucose concentration. The Aminex HPX-87H column (300 mm × 7.8 mm; Bio-Rad, USA) was maintained at 65°C with an eluent that contained 0.01 N, H2SO4.
The nitrate concentration was measured with an ion chromatograph (883 Basic IC Plus, Switzerland) with an anion column (Metrosep A Supp 5). The eluent, which consisted of 3.2 mM Na2CO3 and 1 mM NaHCO3, was supplied at a flow rate of 0.7 ml min-1 into the column for the analysis [23 (link)].
The nitrate concentration was measured with an ion chromatograph (883 Basic IC Plus, Switzerland) with an anion column (Metrosep A Supp 5). The eluent, which consisted of 3.2 mM Na2CO3 and 1 mM NaHCO3, was supplied at a flow rate of 0.7 ml min-1 into the column for the analysis [23 (link)].
5
Quantitative Amino Acid Analysis in Yeast
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Three independent cultures of control and Tfe2-expressing S. cerevisiae cells were incubated for 6 h at 30°C in galactose induction media, washed twice with ice-cold water, normalized to 5 OD600 per 1 ml of water and boiled for 15 min. After centrifugation, the supernatant was clarified using a 0.2 µm syringe filter (Sartorius). The resulting extract was acidified by addition of TFA in MeOH (10%, v/v), dried and resuspended in buffer (40% 1M NaOAc, 40% MeOH, 20% triethylamine, TEA), before being dried again. The amino acids were then derivatized using Phenylisothiocyanate (PITC) by resuspension of the dried material in 10% PITC, 70% MeOH, 10% H2O, 10% TEA, followed by 2-fold dilution in MeOH. After 15 min incubation at room temperature, suspensions were dried, washed once with MeOH and suspended in elution buffer, composed of 95% (150 mM NaOAc, 0.5 ml/l TEA, pH 6.4) and 5% acetonitrile. PTC amino acid derivatives were analysed by HPLC using a Nova-Pak® C18 column (Waters), the Clarity Lite software and two amino acid standard sets (AA-S-18 and A6282, Sigma). The experiment presented has been reproduced on two independent occasions.
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