The GSPD-1 activity of adult C. elegans was assayed spectrophotometrically at 340 nm by the reduction of NADP+ as previously described (Yang et al., 2013 (link)). In brief, staged day 1 adults were harvested from an NGM agar plate by washing with PBS to remove bacteria. The worms were resuspended in extraction buffer (20 mM Tris–HCl, pH 8.0, 3 mM magnesium chloride, 1 mM EDTA, 0.02% β-mercaptoethanol, 1 mM ε-aminocaproic acid and 0.1% Triton X-100). The worm suspension was chilled immediately on ice and homogenized by a pellet pestle motor (Kontes). The crude lysates were centrifuged at 12,000 r.p.m. for 15 min at 4 °C (Z 233 MK-2, Hermle) and the supernatants (protein-containing lysate) were obtained. The Protein concentration of the lysate was determined by the Bradford method (Bio-Rad, Hercules, CA, USA). A typical assay mixture consisted of 100 mg of protein lysate in 0.2 ml of assay buffer (50 mM Tris–HCl pH 8, 50 mM MgCl2, 4 mM NADP+, 4 mM glucose 6-phosphate). The change of absorbance at 340 nm in each sample was measured spectrophotometrically for 15 min at 37 °C (SPECTROstar Nano, BMG Labtech).
Z 233 mk 2
Manufactured by Hermle
Sourced in Germany
The Z 233 MK-2 is a laboratory centrifuge produced by Hermle. It is designed for general-purpose centrifugation applications. The centrifuge features a maximum speed of 15,000 rpm and a maximum RCF of 21,382 x g. It can accommodate a wide range of rotor options to suit different sample types and volumes. The Z 233 MK-2 is a versatile and reliable laboratory equipment piece.
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Lab products found in correlation
Spectrostar nano, by BMG Labtech (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Quercetin, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Carlo Erba (1 mentions)
8453 uv vis spectrophotometer, by Agilent Technologies (1 mentions)
Waters 2410 refractive index detector, by Waters Corporation (1 mentions)
600e system, by Waters Corporation (1 mentions)
Sugar pak 1 ion exchange column, by Waters Corporation (1 mentions)
Waters column heater module, by Waters Corporation (1 mentions)
Model v 530, by Jasco (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
8 protocols using z 233 mk 2
1
Assaying GSPD-1 Activity in C. elegans
2
Tissue Homogenization and Dopamine Quantification
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Tissue homogenization was performed according to Chi et al. [48 (link)] and our previous work [40 (link)]. Briefly, the tissue was collected in 400 μL of 0.2 M perchloric acid and then homogenized in a glass-glass homogenizer in ice. The homogenate was centrifuged at 12,000 ×g for 15 minutes at 4°C (model Z233MK-2, Hermle LaborTechnik GmbH, Wehingen, Germany) and the resultant supernatant was filtered (0.2 μm HPLC Syringe Filters disposable filter PTFE, model EW-32816-26, Cole-Parmer Instrument Company, USA). The filtered supernatant was injected into a HPLC coupled to electrochemical detection for determination of DA content. The pellet was resuspended in 1 N NaOH for protein quantification by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., Richmond, CA, USA) using bovine serum albumin as standard. The DA content was expressed as picograms per milligram of total protein.
3
Ascorbate Determination in Plant Tissue
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Ascorbate determination followed the procedure described by Law et al. (1983) (link). Frozen plant tissue was homogenized with 5 % meta-phosphoric acid at a ratio of 1 : 5 (w/v). The extract was centrifuged at 14 000 g for 20 min at 4 °C (Z 233 MK-2, HERMLE) and the supernatant was collected. Reduced ASC was assayed in 5 mM ethylenediaminetetraacetic acid in 150 mM sodium-phosphate buffer (pH 7.4). Dehydroascorbate (DHA) was reduced to ASC prior to determination by the addition of 10 mM dithiothreitol (DTT), the excess of which was oxidized after 15-min incubation by the addition of 0.5 % N-ethylmalemide. The colorimetric assay was carried out at 37 °C for 1 h after the addition of a mixture containing 10 % trichloroacetic acid, 44 % o-phosphoric acid, 4 % α,α-dipyridil in ethanol and 3 % aqueous ferric chloride solution at a ratio of 2 : 2 : 1 : 1 (v/v). Light absorbance of the resultant samples was measured at 525 nm. Dehydroascorbate was calculated by the difference between total ASC and reduced ASC.
4
Quantification of Flavonoids in Samples
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Flavonoids were measured according to the method outlined by Heimler et al. [67 (link)]. An amount of 500 mg of ground samples was homogenized in 2 mL of 80% ethanol and then centrifuged at 15,000 rpm (Z 233 MK-2, Hermle, LaborTechnik GmbH, Wehingen, Germany) for 5 min. The supernatant (300 µL) was added to 45 µL of a 10% AlCl3 (Carlo Erba, Cornaredo, Milan, Italy) solution, 300 µL of a 1M NaOH (Carlo Erba, Cornaredo, Milan, Italy) solution, and 300 µL of deionized water. Samples were read at 510 nm with a UV-Vis spectrophotometer (8453, Agilent, Santa Clara, CA, USA). Quantification was performed with a calibration curve (5–200 µg mL−1) of quercetin (Sigma-Aldrich, Burlington, MA, USA), and the results were expressed as mg of quercetin equivalent on a dry weight basis (mg QE g−1 dw).
5
Quantification of Soluble Sugars by HPLC
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The soluble sugar content was measured following the method described by Fedeli et al. [69 (link)]. Ground samples of 100 mg were homogenized in 2 mL of deionized water and then centrifuged at 15,000 rpm (Z 233 MK-2, Hermle, LaborTechnik GmbH, Wehingen, Germany) for 5 min. The supernatant was filtered at 0.45 μm using a syringe filter (Lab Logistic Group GmbH, Meckenheim, Germany) and then directly analyzed by an HPLC system (600E System, Waters, Milford, MA, USA) equipped with a Waters 2410 refractive index detector. Sugar separation was allowed using deionized water as mobile phase, eluted at 0.5 mL min−1, and a Waters Sugar-Pak I ion-exchange column (6.5 × 300 mm) kept at 90 °C using an external temperature controller (Waters Column Heater Module, Milford, MA, USA). Quantification of sucrose, glucose, and fructose was obtained through calibration curves prepared by dissolving analytical sugars (Sigma-Aldrich, USA) in deionized water at concentrations of 0.1–20 mg mL−1. The results were expressed on a dry weight basis.
6
Determination of Total Polyphenols
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Total polyphenols were determined according to Singleton and Rossi (1965) . Frozen leaf samples were extracted with 100 mM sodium-phosphate buffer (pH 7) at a 1 : 4 (w/v) ratio using a mortar and pestle. Resulting extracts were centrifuged for 20 min, 14 000 g at 4 °C (Z 233 MK-2, HERMLE). Aliquots of the collected supernatant were assayed with 2 N Folin–Cioclateur reagent (Sigma) and 35 % Na2CO3, by 60-min incubation at 30 °C in a water bath. The absorbance of the resulting colour was measured at 730 nm (JASCO, model V-530) against a known standard of gallic acid. Total polyphenols were expressed in mg 100 g−1 FW as gallic acid equivalents (GAE).
7
DPPH-Based Antioxidant Activity Determination
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The free radical scavenging activity was determined following the method described by Vannini et al. [71 (link)]. About 100 mg of ground samples was homogenized in 2 mL of 80% (v/v) of ethanol and then centrifuged at 15000 rpm (Z 233 MK-2, Hermle, LaborTechnik GmbH, Wehingen, Germany) for 5 min. An aliquot of supernatant (200 µL) was added to 1 mL of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) (Sigma-Aldrich, USA) solution, previously prepared by dissolving 1.85 mg of this compound in 50 mL of 80% methanol (v/v) (Carlo Erba, Cornaredo, Milan, Italy). To compare the antioxidant power of the samples, a blank and a control were prepared by adding 200 µL of 80% (v/v) ethanol (Carlo Erba, Cornaredo, Milan, Italy) in 1 mL of 80% (v/v) methanol (Carlo Erba, Cornaredo, Milan, Italy) and in 1 mL of DPPH solution, respectively. The reaction for all preparations was conducted in the dark for 1 h, and then the absorbance was read at 517 nm with a UV-Vis spectrophotometer (8453, Agilent, Santa Clara, CA, USA). The results were expressed as a percentage of antiradical activity (ARA %), according to the following formula:
8
Green Synthesis of Gold Nanoparticles
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Preparation of Au-NPs was performed (n = 3) following the method described in the literature [18 (link)] with minor modifications using aqueous extracts of ephedra and pistachio leaves as reducing and capping agents. Briefly, 1 mL of the extracts was added to the oxidizing agent of gold (0.005 M HAuCl4) at pH 7.0. The concentration of HAuCl4 was 0.005 M, and the reaction was done at the ratio of 1:1.5 (HAuCl4: extract).
The reaction was carried out at room temperature in an aqueous environment. Changes in the color of the mixture from yellow to pink and then to red was considered as an indication of reaction process. The formation of NPs was monitored by UV-Vis absorption spectrometer (Optima, Tokyo, Japan) using wavelengths of 560, 570, 590, and 610 nm. Upon completion of the reaction, the mixtures were centrifuged (Hermle Z233 MK-2; Hermle, Wehingen, Germany) at 5000 rpm for 10 min to separate the NPs. The collected NPs were then re-suspended in distilled water to remove any impurities. The method was repeated thrice and finally dried in vacuum at 30 °C.
The reaction was carried out at room temperature in an aqueous environment. Changes in the color of the mixture from yellow to pink and then to red was considered as an indication of reaction process. The formation of NPs was monitored by UV-Vis absorption spectrometer (Optima, Tokyo, Japan) using wavelengths of 560, 570, 590, and 610 nm. Upon completion of the reaction, the mixtures were centrifuged (Hermle Z233 MK-2; Hermle, Wehingen, Germany) at 5000 rpm for 10 min to separate the NPs. The collected NPs were then re-suspended in distilled water to remove any impurities. The method was repeated thrice and finally dried in vacuum at 30 °C.
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