For ORAC analysis, 75 mM potassium phosphate buffer (pH 7.4) was used to prepare Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) standards ranging from 0–100 μM, and to dilute the filtered supernatant to a final concentration of 50 μg GAE/mL. After which, 25 μL of samples and standards were loaded into a black 96-well plate, followed by 150 μL of sodium fluorescein working solution (81 nM fluorescein in phosphate buffer). The plate was incubated in the dark for 30 min at 37°C followed by addition of 25 μL of 2,2’-azobis (2-amidinopropane) dihydrochloride solution (152 mM in the phosphate buffer). A fluorescence spectrophotometer (Synergy microplate reader) was used to monitor fluorescence decay at 37°C, 485/20 nm excitation and 528/20 nm emission wavelengths, every one min for 120 min or until less than 0.5% of the initial value was reached. After subtraction of the blank, the area under the curve (AUC) for the standard curve and the samples were used for calculations. The results were expressed as μmol of Trolox per g fresh fruit (μmol TE/ g sample).
Synergy microplate reader
Manufactured by Synergy Software
The Synergy microplate reader is a versatile instrument designed for performing a wide range of absorbance, fluorescence, and luminescence-based assays. It features a compact and modular design, offering flexibility to accommodate various microplate formats and detection modes. The Synergy microplate reader is capable of accurately measuring optical signals from samples in microplates, supporting researchers in a variety of applications.
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Lab products found in correlation
7 protocols using synergy microplate reader
1
Assessing Antioxidant Capacity via ORAC
2
Evaluating sRNA Effects on Growth
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To assess the effects of manipulating levels of selected sRNAs on growth rate, overnight cultures of mutant and wildtype strains were inoculated (1:100) into MS-hiC and MS-loC media. All samples were grown at RT with intermittent shaking and absorbance (OD = 600 nm) measurements were recorded every 30 min for 16 h. All measurements were taken on the same Synergy microplate reader as above.
3
Antioxidant Capacity Measurement by ORAC
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ORAC was measured as described [26] (link) with slight modifications. All reagents and sample dilutions were made in 75 mM PBS, pH 7.4. Stock solutions of fluorescein 1 mM and Trolox 2.5 mM were prepared and single use aliquots were stored at −20°C. Water was pipette into the outer wells of a 96-well plate. Fluorescein was diluted to 6 nM from the stock solution and 150 µL of was added to each well. Next, 25 µL of Trolox standard (0–50 µM) or samples were added to each well in triplicate. The plate was pre-incubated at 37°C for 30 min. A fresh solution of AAPH was prepared and 25 µL added to each well after pre-incubation to a final concentration of 16 mM. Fluorescence readings were recorded every minute for 75 min on the Synergy microplate reader (485 nm, 20 nm bandpass, excitation filter and a 528 nm, 20 nm bandpass, emission filter). A regression line was generated by plotting the concentration of Trolox by net area under the curve (AUC). Results were expressed as µmol Trolox equivalents (TE) ± SE.
Additionally, lyophilized leaves were sent to Brunswick laboratories (Southborough, MA) for external validation of antioxidant capacity by ORAC-H [27] , [28] (link).
Additionally, lyophilized leaves were sent to Brunswick laboratories (Southborough, MA) for external validation of antioxidant capacity by ORAC-H [27] , [28] (link).
4
Quantitative DPP4 Activity Assay
5
Calpain Activity Assay Protocol
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Cells (7 × 106/ml) were rinsed, scraped, and homogenized in
extraction buffer (10 mM Tris-HCl, pH 7.5). The homogenate was then centrifuged at
14,000 rpm for 10 min, and 50 to 100 μl of the supernatant, or purified
calpain was assayed for activity. The reaction mixture included (final
concentrations) 200 mM Tris-HCl, pH 8.0, 20 mM CaCl2, 10 mM cysteine and
50 μM Suc-LYAMC (Sigma) as substrate. Activity was monitored as the change in
fluorescence as a function of time measured at excitation/emission wavelengths of 345
nm and 445, respectively, in a 96-well plate using a Synergy microplate reader.
Purified calpain and its activity was measured using a calpain activity assay kit
(ab65308) as described in the manufacturer's instructions.
extraction buffer (10 mM Tris-HCl, pH 7.5). The homogenate was then centrifuged at
14,000 rpm for 10 min, and 50 to 100 μl of the supernatant, or purified
calpain was assayed for activity. The reaction mixture included (final
concentrations) 200 mM Tris-HCl, pH 8.0, 20 mM CaCl2, 10 mM cysteine and
50 μM Suc-LYAMC (Sigma) as substrate. Activity was monitored as the change in
fluorescence as a function of time measured at excitation/emission wavelengths of 345
nm and 445, respectively, in a 96-well plate using a Synergy microplate reader.
Purified calpain and its activity was measured using a calpain activity assay kit
(ab65308) as described in the manufacturer's instructions.
6
Glutathione Reductase Activity Quantification
7
Disaggregase ATPase Activity Characterization
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Steady-state ATPase activity was measured in 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 20 mM MgCl2 at 25 °C, as previously described62 (link) in the presence of an ATP-regenerating system29 (link). Final ClpB and ATP concentrations were 2 μM and 2 mM, respectively. The activity of the same samples was also measured in the presence of 3.5 μM DnaK, 0.7 μM DnaJ, and 0.35 μM GrpE, to study the DnaK-induced activation of the disaggregase. Substrate-induced stimulation of the ATPase activity was determined in the presence of 0.4–1.6 μM G6PDH aggregates formed at 50 or 80 °C, 10 μM α-casein, 5 µM RepE, 10 µM RCMLA, 10 μM F-APPY, or 350 μM NR peptide (NRLLLTG). Reactions were followed by measuring the absorbance decay at 340 nm in a Synergy microplate reader using 96-well plates, at 25 °C. The concentration of all the substrates was saturating, except that of protein aggregates. The scattering of these samples at higher concentrations precluded the use of the coupled assay employed in these measurements.
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