DNA binding was analyzed by fluorescence anisotropy employing a splayed duplex DNA with a cy3 label Cy3-5′- AGCTACCATGCCTGCACGAATTAAGCAATTCGTAATCATGGTCATAGC-3′ and 5′-GCTATGACCATGATTACGAATTGCTTGGAATCCTGACGAACTGTAG-3′ (red color denotes ssDNA region). Assays were carried out in 20 mM HEPES pH 7.5, 50 mM KCl, 5 mM MgCl2, 1 mM TCEP, and 5 nM DNA at room temperature. Wild-type ctXPD and its variants were used at concentrations of 2–2000 nM as indicated. After mixing, the reaction was incubated for 5 min prior to recording. Fluorescence was detected at an excitation wavelength of 540 nm and an emission wavelength of 590 nm with a Floustar Optima plate reader (BMG labtech). The gain was adjusted to a well containing buffer and DNA but no protein. Curves were fitted with Graphpad Prism and represent the averages of at least three different reactions and two independent protein batches and mean values are plotted with their associated SD.
Floustar optima plate reader
Manufactured by BMG Labtech
Sourced in Germany
The FLUOstar Optima is a fully-automated, high-performance microplate reader designed for a wide range of fluorescence and luminescence-based assays. It features a reliable, sensitive detection system and can be configured with a variety of optical modules to meet specific research needs.
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5 protocols using floustar optima plate reader
1
DNA Binding Assay by Fluorescence Anisotropy
2
Cell Growth Kinetics Assay
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Cell growth was determined using CellTiter-Glo Luminescent Cell Viability Assay reagent (Promega) following manufacturer’s instructions. Fibroblast cells were plated on 96-well plates at 1,000 cells per well and cultured in complete medium. Proliferation was measured every 24 hr for 120 hr and therefore at six time points in total. Cells were lysed using freshly prepared assay reagent and incubated for 10 min at each end point. Cell lysates were then transferred to white flat-bottom plate for luminescence measurement on a FLOUstar Optima plate reader (BMG labtech). Cell growth was calculated relative to time 0 for each cell.
3
Amyloid Fibrillation Kinetics Monitoring
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αSN was fibrillated in 96 well clear bottom plates (NUNC) on a Floustar Optima Platereader (BMG labtech). Fibrillation was performed in triplicates at 12 mg/ml with 20 uM Thioflavin T (ThT) (Sigma Aldrich) as fluorescent marker and a sample volume of 150 µL per well. The assays were conducted using same conditions used in Giehm et al.66 (link) including heating (37°C), shaking (orbital shaking, 300 rpm, 2 mm) in cycles of 280 sec followed by 80 sec pause. Each well contained a 3 mm glass bead to increase reproducibility.35 (link) ThT fluorescence was employed as an internal clock of the progress of protein amyloid fibrillation.67,68 (link) The emission was recorded at 480±5 nm after excitation at 450±5 nm.
4
Quantifying Circulating Biomarkers in Diabetes
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Circulating levels of sCD163, sST2 and Gal-3 were investigated in plasma using commercially available DuoSet ELISA kits (#DY1607, #DY523B-05 and #DY1154) and supplementary ancillary kit (#DY008; R&D Systems, Minneapolis, MN, USA). Participant plasma samples were diluted 1:200, 1:20 or 1:2 in PBS with 1% BSA for sCD163, sST2 and Gal-3 respectively. The diluted samples were run in duplicates and subgroups were alternated on each ELISA plate to minimize bias caused by intra- and inter-variations. The ELISA analyses were run in accordance with the manufacturer’s instructions. Absorbance was measured at 450 nm using a FLOUstar Optima plate reader (BMG Labtech Gmbh, Ortenburg, Germany). Sample concentrations were predicted by plotting a 4-parameter fit using a 7-point standard curve. The intra- and inter-coefficient of variation reached 1.9% and 5.1%, 3.9%, and 6.0%, and 3.1% and 12.4% for sCD163, sST2 and Gal-3 respectively.
HbA1c values at type 1 diabetes diagnosis were analyzed in Capillarys TERA Hemoglobin A1c Kits [24 (link)] and thereafter in Alere Afinion AS100 analyzer [25 (link)]. For HbA1c IFCC-units mmol/mol were used.
HbA1c values at type 1 diabetes diagnosis were analyzed in Capillarys TERA Hemoglobin A1c Kits [24 (link)] and thereafter in Alere Afinion AS100 analyzer [25 (link)]. For HbA1c IFCC-units mmol/mol were used.
5
Quantifying IL-6 Secretion by ELISA
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Culture supernatants were harvested following the treatments and assayed for IL-6 levels by ELISA assay (Quantikine® ELISA, RnDSystems, Abingdon, UK) according to the manufacturer's instructions. Absorbance was recorded at 450 nm and corrected to 570 nm using a FLOUstar OPTIMA plate reader (BMG Labtech, Ortenberg, Germany). The IL-6 concentration was calculated from a standard curve for all experiments. Data is represented as the fold change in IL-6 concentration in the supernatant between control and treated cells.
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