LLE-fluor and WSR-rhod at 20 μM were added to varying concentrations of HIV-1 PR in the PR assay buffer described above, adjusted to pH 6 to a final volume of 60 μL in the wells of a 384 clear-bottom black microwell plate. The plate was incubated in the dark at 4 °C for an hour before the fluorescent spectra were read on a SpectraMax M5 plate reader (Molecular Devices). The solution was excited at 480 nm, and the emission was read between 510 and 600 nm with a cutoff value of 495 nm.
Spectramax m5 plate
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax M5 plate reader is a versatile multi-mode microplate reader capable of absorbance, fluorescence, and luminescence detection. It features a unique, patented dual-mode optics design for enhanced sensitivity and flexibility. The SpectraMax M5 plate reader can be used for a wide range of applications in life science research, drug discovery, and quality control.
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Lab products found in correlation
5 protocols using spectramax m5 plate
1
Fluorescent Assay for HIV-1 Protease
2
Quantification of Inflammatory Cytokines
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Cortex tissue from all treatment groups was homogenized using a Miltenyi Biotec gentleMACS™ Dissociator and total protein was quantified using a ThermoFisher Pierce™ BCA Protein Assay kit. Capture antibodies against CCL2, IL-6, or TNFα were incubated overnight in 96-well plates. Each ELISA was performed according to the instructions provided from R&D Systems and the total input protein for each sample was 100 μg or serum diluted 1:10. Absorbance was read using a SpectraMax® M5 plate reader from Molecular Devices (Sunnyvale, CA, USA). Data were reported as CCL2, IL-6 or TNFα concentration per mg of total lysate protein or per ml of serum.
3
Peptide Demodification Assay Analysis
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The peptide demodification assay was described earlier47 (link). Briefly, peptide concentration for the assay were estimated using absorbance at λ260nm with a molar extinction coefficient of 13,400 M−1 cm−1 for the ADP-ribosyl modification. 8 μM of indicated peptide was demodified by incubation with 1 μM indicated hydrolase (or various concentrations of MavL/Larg1) for 30 min at 30 °C in assay buffer (50 mM Tris-HCl pH 8, 200 mM NaCl, 5 mM MgCl2, 1 mM DTT and 0.15 μM human NUDT5)65 (link). Reactions were stopped and analyzed by performing the AMP-Glo™ assay (Promega) according to the manufacturer’s protocol. Luminescence was recorded on a SpectraMax M5 plate reader (Molecular Devices) and data analyzed with GraphPad Prism 7. For background subtraction reaction were carried out in absence of hydrolase.
4
High-Throughput Protein Binding Assay
5
Antioxidant and ROS Assays in ARPE-19 Cells
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The GSH level, superoxide dismutase (SOD) activity, and GPx activity were determined using commercial assay kits [51 (link)]. Briefly, cells were placed in 6-well plates at a density of 5 × 105 cells per well. After 12 h, the cells were treated for 48 h with 400 μM of COS–5 or N–5 and then for 24 h with or without 75 μM acrolein. After treatment, the cells were washed twice with PBS, and then the antioxidant enzyme activities and GSH level in the cells were detected.
Moreover, the ROS levels in PRE cells and mitochondria exposed to acrolein were determined using fluorescent probe. In brief, cells were plated in 96-well plates at a density of 2.5 × 104 cells per well for 12 h. ARPE-19 cells were treated with 400 μM of COS–5 or N–5 for 48 h, and then incubated with or without 75 μM acrolein for another 24 h. The ROS level in PRE cells was determined by the 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) method using a SpectraMax M5 plate reader (Molecular Devices, San Jose, CA, USA) at a 488 nm excitation wavelength and a 525 nm emission wavelength [52 (link)]. The ROS generation in mitochondria was detected using MitoTracker Red CM-H2Xros at a 579 nm excitation wavelength and a 599 nm emission wavelength.
Moreover, the ROS levels in PRE cells and mitochondria exposed to acrolein were determined using fluorescent probe. In brief, cells were plated in 96-well plates at a density of 2.5 × 104 cells per well for 12 h. ARPE-19 cells were treated with 400 μM of COS–5 or N–5 for 48 h, and then incubated with or without 75 μM acrolein for another 24 h. The ROS level in PRE cells was determined by the 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) method using a SpectraMax M5 plate reader (Molecular Devices, San Jose, CA, USA) at a 488 nm excitation wavelength and a 525 nm emission wavelength [52 (link)]. The ROS generation in mitochondria was detected using MitoTracker Red CM-H2Xros at a 579 nm excitation wavelength and a 599 nm emission wavelength.
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