Peptides were tested using DPPH technique to estimate their hypothetical antioxidant activity. The assay was performed according to the methodology described by [27 (link)]. Briefly, 198 µL of the DPPH solution (0.1 mM) was mixed with 2 µL of sample solution (10 µM). Then, the mixture was incubated in the dark for 30 min at room temperature. Absorbance was measured at 517 nm using a microplate reader (Bioteck, Epoch/2 microplate reader, Winooski, VT, USA). The results were expressed in percentage of control.
Epoch 2 microplate reader
Manufactured by Bioteck
Sourced in United States
The Epoch/2 microplate reader is a versatile instrument designed for various absorbance-based assays. It can measure the optical density of samples in a microplate format, enabling high-throughput analysis. The Epoch/2 provides accurate and reliable data for a wide range of applications.
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4 protocols using epoch 2 microplate reader
1
Peptide Antioxidant Potential via DPPH Assay
2
Neuroprotective Effects of Compound 1
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Effects on cell viability and neuroprotection assays were estimated using the MTT (VWR, Solon, Ohio, USA) method, as described by Silva et al. [22 (link)]. SH-SY5Y cells were exposed 24 h to 6-OHDA (100 µM) in absence (Vehicle) or presence of compound 1 at concentrations of 1–100 μM. Then, 100 µL MTT (1.2 mM) were added to wells and cells were incubated for 1 h at 37 °C. After this time, MTT was removed and 100 µL DMSO was added. The resulting absorbance was read in a microplate reader (Bioteck, Epoch/2 microplate reader, Winooski, VT, USA) at 570 nm. The results were expressed in percentage of control.
3
Ferric Reducing Ability of Peptides
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FRAP assay adapted to microscale with minor modifications [27 (link)] was used to measure the ferric reducing ability of peptides. Acetate buffer (0.3 M, pH 3.6), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ, 10 mM prepared in 40 mM HCl), and ferric solutions (20 mM) were freshly mixed at a ratio of 10:1:1 and place in a 96-well microplate. Then, 198 µL of FRAP reagent and 2 µL of the peptide sample was mixed and incubated at 37 ºC in the dark for 30 min. The absorbance was measured at 593 nm using a microplate reader (Bioteck, Epoch/2 microplate reader, Winooski, VT, USA). The difference between the absorbance of the test sample and the blank reading was calculated and expressed as µM of FeSO4/g of the peptide. FeSO4 was used as standard.
4
SARS-CoV-2 RBD Antibody Detection Assay
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To perform the assay, a fixative solution containing 1 µg/mL of SARS-CoV-2 RBD protein (GenScript) was prepared in carbonate-bicarbonate buffer (pH 9.6) and then a Nunc MaxiSorp flat bottom plate (Sigma) was coated with 100 µL of the fixative solution and incubated at 4°C overnight. The next day, the plate was washed five times with DPBS 0.05% (v/v) Tween-20 buffer (0.05% DPBS-T) and blocked with 3% (w/v) of skim milk (BD Biosciences) in 0.05% DPBS-T for 2 hours at room temperature, the plate was then washed five times with 0.05% DPBS-T. 100 µL of sera diluted 1/2000 (week 1 post-vaccination to week 7 post-vaccination) and 100 µL of purified total IgY antibodies (0.3 mg/mL) diluted 1/800 (week 1 post-vaccination to week 10 post-vaccination) both with 1% (w/v) of skim milk were added to the plate and incubated for 1 hour at 37°C. Later, wells were washed five times with 0.05% DPBS-T and immediately incubated with 100 µL of Goat anti-Chicken IgY secondary antibody conjugated with HRP (Genscript) diluted 1/2000 in 1% non-fat milk in 0.05% DPBS-T for 1 hour at 37°C. The plate was then washed five times with 0.05% DPBS-T and incubated with 100 µL of 3,3’,5,5’-tetramethylbenzidine (TMB) for 15 min at room temperature. The reaction was stopped by the addition of 50 µL of 2N H2SO4 per well, and the plate was read at 450 nm using an Epoch 2 microplate reader (Bioteck).
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