Total direct microscopic counts (TDC) of bacteria were determined using ethidium bromide with fluorogenic dye as described previously (49 ). In brief, triplicate membrane filters were prepared and bacteria were counted in at least 50 randomly selected microscopic fields of each filter preparation. Culturable bacteria were enumerated on full-strength nutrient broth (NB) and 1:100 diluted nutrient broth (DNB) as the plating agar medium (38 (link)). Four replicates of sample dilutions were plated and incubated at 30°C for 28 days. Fungal propagules were counted on rose bengal agar medium (43 ) in four replicates. Ergosterol was determined as an indicator of fungal biomass by the method of vibration-assisted extraction followed by HPLC quantification (12 , 62 (link)). The HPLC system (Tosoh, Tokyo, Japan) was essentially the same as described previously (62 (link)). A soil sample taken at a forest site unaffected by the 2000 eruption on Miyake-jima and two agricultural soils from the Field Science Center, Ibaraki University College of Agriculture were used as references.
Hplc system
Manufactured by Tosoh
Sourced in Japan
The HPLC system is a high-performance liquid chromatography instrument used for the separation, identification, and quantification of chemical compounds in a liquid mixture. It consists of a solvent delivery system, a sample injection device, a chromatographic column, and a detection system. The HPLC system allows for the precise and efficient analysis of a wide range of substances, including pharmaceuticals, environmental pollutants, and food components.
Automatically generated - may contain errors
Lab products found in correlation
Ods 100v column, by Tosoh (1 mentions)
Amide 80 column, by Tosoh (1 mentions)
Tskgel polyaminepak column, by Tosoh (1 mentions)
Protein assay bca kit, by Nacalai Tesque (1 mentions)
Tsk gel g3000swxl 7.8 300 mm column, by Tosoh (1 mentions)
Amicon ultra 0.5 ml centrifugal filter, by Merck Group (1 mentions)
Tskgel g3000swxl, by Tosoh (1 mentions)
Vitros 950, by Ortho Clinical Diagnostics (1 mentions)
6 protocols using hplc system
1
Enumerating Soil Microbes and Fungal Biomass
2
Purification of Saccharide 1 from Super Ohtaka
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Super Ohtaka® (1 kg, Brix: 59 %) was loaded onto a 4.5 × 35 cm carbon-Celite column consisting of 1:1 charcoal/Celite-535 (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and successively eluted in water (14 L) and 5 % ethanol (30 L). Almost all of glucose and fructose were eluted with water (0–4 L), while the fraction containing the target saccharide named saccharide 1 was eluted in 5 % ethanol fraction (1–3 L). Then, the fraction (2 L) containing the saccharide was concentrated to 10 mL. Subsequently, the concentrated fraction was repeatedly applied to preparative-HPLC (Tosoh Corporation, Tokyo, Japan) equipped with an Amide-80 column (7.8 mm × 30 cm, Tosoh Corporation) at 80 °C and eluted with 80 % acetonitrile at 2 mL/min using refractive index detection (Fig. 1 ). Furthermore, saccharide 1 was purified at room temperature using an HPLC system (Tosoh Corporation) equipped with an ODS-100V column (4.6 mm × 25 cm, Tosoh Corporation) and eluted with distilled water at 0.5 mL/min using refractive index detection. The purified saccharide 1 solution was freeze-dried to give a white powder (1.8 mg).
3
Polyamine Content Quantification Protocol
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Polyamine contents in cells were determined using the method described by Igarashi K et al.29 (link). Cells were washed three times with ice-cold phosphate buffered saline (PBS), homogenized in 10 mM Tris-HCl, pH 8.0 and incubated in 0.2 M perchloric acid at 70 °C for 30 min. After centrifugation, supernatants were collected and resulting pellet were homogenized and incubated in 0.2 M perchloric acid at 70 °C for 30 min. After centrifugation, supernatants were combined and analyzed using a SHIMADZU HPLC system with TSKgel Polyaminepak column (TOSOH, Japan). Protein contents in cells were determined with a BCA protein assay kit (Nacalai tesque) using bovine serum albumin as a standard.
4
SEC Analysis of Protein Samples
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SEC was carried out with a TSKgel G3000 SWXL, 7.8 × 300 mm column (Tosoh) connected to a HPLC system (Tosoh). The isocratic elution was carried out with a mobile phase 0.1 M sodium phosphate, 0.1 M sodium sulphate, 0.05% sodium azide pH 6.7 at a flow rate of 1.0 mL/min. The column temperature was kept at 30 °C during analysis. Fifty µL of each 200 µg/mL sample was injected onto the column and analysed with UV detection at the wavelength of 280 nm.
5
Optimizing hGH Refolding from Inclusion Bodies
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hGH IBs were solubilized in three different ways: (a) 10 μl of hGH IBs were solubilized in 89 μl of 2 M urea and 1 μl of 100 mM DTT, (b) 10 μl of hGH IBs were solubilized in 90 μl of 2 M urea, and (c) 10 μl of hGH IBs were solubilized in 90 μl of 8 M urea. These three suspensions were frozen overnight at −20°C and thawed at room temperature the next morning. Suspensions were centrifuged at 12,000 rpm, 4°C for 30 min. Each of the supernatants was diluted in a pulsatile manner in 900 μl of refolding buffer at 4°C containing 20 mM Tris–HCl. Refolded protein was centrifuged at 12,000 rpm, 4°C for 30 min. All these three protein samples along with commercial hGH were concentrated to 6 mg/ml using concentrators (Amicon® Ultra 0.5 ml Centrifugal Filters, Merck, United States). Concentrated samples were centrifuged again at 12,000 rpm, 4°C for 10 min. Twenty microliters of each sample was loaded onto pre-equilibrated (20 mM Tris–HCl and 100 mM NaCl) HPLC column TSK gel G3000SWxl (Tosoh Biosciences, Japan) using the Waters HPLC system. The graphs of different protein samples were overlaid and the area under the curve was determined.
6
Assessing Diabetes Risk Factors
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The primary cross-sectional outcomes in this analysis were fasting glucose, homeostatic model assessments of beta cell function (HOMA-β) and insulin resistance (HOMA-IR), and hemoglobin A1c (A1c). Fasting glucose and insulin concentrations were measured on a Vitros 950 or 250 Ortho-Clinical Diagnostics analyzer using standard procedures that met the College of American Pathologists accreditation requirement (Carpenter et al., 2004 (link)). A HPLC system (Tosoh Corp) was used to measure A1c. Insulin resistance and β-cell function were estimated using the following formula: HOMA-IR = (fasting plasma glucose [millimoles per liter] × fasting plasma insulin [milliunits per milliliter]) / 22.5 and HOMA-β = (20 × fasting plasma insulin) / (fasting plasma glucose − 3.5)% (Matthews et al., 1985 (link)). The primary longitudinal outcome was incident diabetes developed by Exam 3 among those free of diabetes at Exam 1. Diabetes was defined as HbA1c ≥ 6.5 % (48 mmol/mol), fasting blood glucose ≥ 126 mg/dL, taking T2DM medications and/or with a self-reported physician diagnosis (American Diabetes Association, 2013 (link)). The time of incident diabetes was defined as the midpoint between the last examination without diabetes and the examination at which diabetes developed among persons without diabetes at baseline.
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