Culture supernatants of WT or mutant Pc Cel6A (10 µL) were incubated with 100 µg of PASC or cellulose III I prepared as described previously 12) 13) in 100 mM sodium acetate buffer (pH 5.0) with a total volume of 200 µL using 96-well plates at 50 or 60 °C with shaking at 1,000 rpm. After two hours of incubation, the solutions were filtered using 96-well plates with a 0.22 µm filter (MultiScreen ® Filter Plates, Merck Millipore, MA, USA). Then, 5 µL of 40 U/mL Aspergillus niger β-glucosidase (Megazyme Ltd.) was added to 100 µL of filtrate, and the plates were incubated at 60 °C for 48 h with shaking at 1,000 rpm. After hydrolysis, the solutions were heated at 98 °C for 3 min and filtered again. The concentration of glucose in filtrate was quantified using Glucose CII-Test Wako (FUJIFILM Wako Pure Chemical Corporation) by measuring the absorbance at 492 nm with a Thermo Scientific Multiscan ® FC (Thermo Fisher Scientific).
Thermo scientific multiscan fc
Manufactured by Thermo Fisher Scientific
The Thermo Scientific Multiscan® FC is a microplate reader designed for a variety of absorbance-based assays. It offers fast and accurate measurements across a wide range of wavelengths.
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Lab products found in correlation
2 protocols using thermo scientific multiscan fc
1
Enzymatic Hydrolysis of Cellulose Substrates
2
Phenoloxidase Enzyme Activity Assay
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For the determination of phenoloxidase enzyme activity, 20 l hemolymph leaking from the anterior segment of the prolegs through the hole opened with a sterile needle were collected from each Pyrethrum injected sample. The collected hemolymph fluid was then placed in microcentrifuge tubes containing 180 l phosphate buffer solution ice-cold and immediately frozen at -20 °C without allowing it to darken. This hemolymph-phosphate buffer mixture, which was dissolved before the experiment, was centrifuged at 10,000 g for 5 minutes in a refrigerated centrifuge (Hettich, Germany) at +4 °C and the supernatant was collected. 40 l of this supernatant was taken and placed in a 96-well microplate. Then, 160 l 3,4-Dihydroxy-Lphenylalanine (L-DOPA-Sigma-Aldrich, St Louis, MO) dissolved in phosphate buffer solution at a rate of 3 mg/ml was added onto the microplate. The prepared microplate was read in ELISA microplate reader (ThermoScientific Multiscan FC) at 490 nm (A490) absorbance at intervals of 5 minutes from 0 to 30 minutes. The data obtained for each subject was determined as U/mg protein/min (Brookman et al., 1989) .
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