Glutathione coated plate (Pierce Biotechnology, USA) was incubated with 100 μl/well of GST-NS1 (100 nM in phosphate-buffered saline [PBS]) for 1 h at room temperature. The wells were blocked with 5% BSA in PBS with Tween 20 (PBST) for 1 h, and followed by incubation with various concentrations of aptamers (100 μl/well in PBS) for another 1 h at room temperature. Unbound GST-NS1 proteins were detected by incubating with 100 μl/well of NS1 antibody (1:1,000 in PBS; Santa Cruz Biotechnology, USA) and 100 μl/well of goat-anti-mouse-horseradish peroxidase (HRP) (1:1,000 in PBS; Santa Cruz Biotechnology) for 1 h at room temperature. After washing, 100 μl/well of 3,3′,5,5′-tetrametylbenzidine (TMB) solution (Merck, Germany) was added to detect the bound HRP. The color developing reaction was stopped with 0.5 N H2SO4 and the absorbance of each well was measured at 450 nm using a TRIAD microplate reader (Dynex Technologies, USA). The percentage of inhibition (Y axis) was calculated from the following equation: inhibition percentage = 100 – [(Abs/A0) × 100], where Abs is the absorbance in the presence of aptamer and A0 is the absorbance in the absence of aptamer.
Triad microplate reader
Manufactured by Dynex
Sourced in United States
The TRIAD microplate reader is a versatile and compact instrument designed for a range of photometric measurements. It can perform absorbance, fluorescence, and luminescence detection in 96-well microplates. The TRIAD offers a robust optical system and user-friendly software to facilitate efficient data collection and analysis.
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Lab products found in correlation
2 protocols using triad microplate reader
1
Glutathione-based Aptamer Screening Assay
2
Aptamer-Based Detection of HA1 Protein
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Aptamers were 5′-biotinylated by asymmetric PCR using the forward primer 5′-Biotin-GCAATGTACGGTACTTCC-3′ followed by lambda exonuclease digestion, as previously described [27 (link),28 (link)]. The 5′-biotinylated ssDNA aptamers (100 nM) were heated at 90°C for 10 min, immediately placed on ice, added to the wells of a streptavidin-coated plate (Pierce Biotechnology, Rockford, IL), and incubated for 1 h at room temperature while shaking at 100 rpm. The wells were washed four times with PBST (0.1% Tween 20 in PBS; pH 7.4), blocked with 5% BSA in PBST at room temperature for 1 h, re-washed four times, and incubated with various concentrations of purified GST-H1-HA1 in PBS at room temperature for 1 h. After washing four times with PBST, incubation with GST antibody-conjugated horseradish peroxidase (HRP; 1:1,000 in PBST, Santa Cruz Biotechnology, Dallas, TX) at room temperature for 1 h, and four additional washes, bound GST-tagged HA1 protein was detected by adding 3,3′,5,5′-Tetramethylbenzidine (TMB) solution (Merck, Darmstadt, Germany) and terminating with 0.5 N H2SO4. The absorbance of each well was measured at 450 nm by using a TRIAD microplate reader (Dynex Technologies, Chantilly, VA). GST-H5-HA1 and GST served as negative controls.
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