Hemolymph of WT and BmTorso mutants at wandering and pupal stages respectively were collected. The samples were extracted with methanol as previously described (Warren et al., 2006 (link)). The extracts were evaporated and re‐dissolved in enzyme immunoassay (EIA) buffer (Cayman), and EIA kit (Cayman Chemical, MI, USA) as described before (Zeng et al., 2017 (link)). Anti‐20‐hydroxyecdysone EIA antiserum, acetylcholinesterase (AchE)‐conjugated 20E, and standard 20E (Sigma) were used in competitive assay to quantify 20E titers. The AchE activity was quantified by Ellman's buffer and measured at 405 nm by using a Multiskan FC microplate photometer (Thermo).
Standard 20e
Manufactured by Merck Group
Sourced in United States
The Standard 20E is a compact and versatile laboratory incubator designed for a wide range of applications. It features a temperature range from ambient +5°C to 60°C and a digital temperature control system for precise temperature regulation. The incubator offers a capacity of 20 liters and is intended for use in laboratories, research institutions, and educational settings.
Automatically generated - may contain errors
Lab products found in correlation
Eia kit, by Cayman Chemical (2 mentions)
Ellman s reagent, by Cayman Chemical (2 mentions)
Multiskan fc, by Thermo Fisher Scientific (2 mentions)
Thermo varioskan flash, by Thermo Fisher Scientific (1 mentions)
Benchmark microplate reader, by Bio-Rad (1 mentions)
Eia buffer, by Cayman Chemical (1 mentions)
20e eia antiserum, by Cayman Chemical (1 mentions)
20e ache tracer, by Cayman Chemical (1 mentions)
5 protocols using standard 20e
1
Ecdysteroid Quantification in Hemolymph
2
Quantification of 20E in Silkworms
3
Quantitative Analysis of 20E in Shoot Cultures
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Control and treated shoots were used for 20E estimation. HPTLC was performed using Muchate et al. method28 (link). The shoot culture samples were powdered in liquid nitrogen. Dry powder (1 g) of each sample was suspended in ethanol (3 ml) and kept at room temperature for 12 hr. Then, the samples were centrifuge at 10,000 rpm (revolution per minute) and supernatant was filtered through 0.22 µM filter. The filtrate was used for analysis. For preparation of standard stock solution, 2.15 mg of standard 20E (purity93%) (Sigma-Aldrich) was dissolved in 2 ml of ethanol (99.9%). The 50 µl of stock solution was diluted by addition of 950 µl of ethanol and used as working standard solution (20E concentration 1 µl = 50 ng) for analysis of 20E using HPTLC.
The HPTLC plate was rinsed with ethanol-ethyl acetate-water (8: 2: 0.5, v/v/v) and then, spots were loaded with various concentrations of standard (50,100, 200, 300, 400, and 500 ng per spot) and experimental samples of 20E. For development of plate, the mixture of chloroform-methanol-benzene (12.5: 2.5: 1.5, v/v/v) was used as a solvent system. The dried developed plate was analyzed for the content of 20E in each spot using TLC scanner based on fluorescent quenching at 254 nm.
The HPTLC plate was rinsed with ethanol-ethyl acetate-water (8: 2: 0.5, v/v/v) and then, spots were loaded with various concentrations of standard (50,100, 200, 300, 400, and 500 ng per spot) and experimental samples of 20E. For development of plate, the mixture of chloroform-methanol-benzene (12.5: 2.5: 1.5, v/v/v) was used as a solvent system. The dried developed plate was analyzed for the content of 20E in each spot using TLC scanner based on fluorescent quenching at 254 nm.
4
Quantification of Ecdysteroid Levels
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Total 20Ewere quantified by enzyme immunoassay (EIA). Newly molted sixth instar larvae treated with 2-TD (twenty five larvae/tube with four replicates) were homogenized and extracted as described previously [46 (link)]. The extracts were evaporated, redissolved, and subjected to ecdysteroid enzyme-linked immunosorbent assay (ELISA). The ELISA was performed in a competitive assay format using anti-20E rabbit antiserum (Cayman Chemical, Michigan, USA), acetylcholinesterase-conjugated 20E (Cayman Chemical, Michigan, USA), and standard 20E (Sigma-Aldrich, St. Louis, MO, USA). The acetylcholinesterase activity was quantified by Ellman’s Reagent (Cayman Chemical, Michigan, USA), and the absorbance at 415 nm was detected with a Benchmark microplate reader (Bio-Rad Laboratories, Hercules, USA).
5
Ecdysteroid Quantification in Larvae
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Ten larvae were rinsed with distilled water, and collected in a 1.5 ml microcentrifuge tube. The larvae were homogenized in 400 μl of methanol with a plastic pestle at room temperature. The samples were centrifuged at 15,000 g for 5 min at 4°C, and 60 μl of the supernatant (equivalent to 1.5 larvae) was subjected to vacuum desiccation. Dried extract was re-dissolved in 50 μl of EIA buffer (Cayman Chemical). Ecdysteroid was quantitated by enzyme-linked immunosorbent assay (ELISA) using 20E EIA antiserum, 20E AchE tracer, and Ellman’s reagent (Cayman Chemical) according to manufacturer’s protocol. Standard 20E was purchased from Sigma.
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