Recombinant proteins (200 ng) diluted in coating buffer (SeraCare, Milford, MA) were incubated in a 96-well microtiter plate (NUNC, Rochester, NY) overnight at 4°C. Then, the plate was blocked using 5% bovine serum albumin in phosphate-buffered saline (PBS) and incubated 1 h at room temperature, and subsequently washed four times in selection buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM MgCl2, 5 mM KCl). The modified apMNK2F aptamers (apMNK2FT and apMNKQ2), conjugated with biotin, were diluted in selection buffer at the concentrations indicated in the figures, denatured for 10 min at 95°C and cooled for 10 min on ice. Next, 100 μL of the diluted aptamers were added to each well, the plate was incubated at 37°C for 1 h and washed four times with selection buffer to remove unbound ssDNA. Afterward, 100 μL of a 1/1,000 dilution of horseradish peroxidase-conjugated streptavidin (GE Healthcare, Madrid, Spain) were added to the individual wells. After 1 h incubation at 37°C on a shaking platform, the plates were washed four times and developed using ABTS solution (Roche, Madrid, Spain) according to the manufacturer’s instructions. OD 405 nm values were determined using a SpectraFluor microplate reader (TECAN, Barcelona, Spain).
Coating buffer
Manufactured by LGC
Sourced in United States
Coating buffer is a solution used to prepare surfaces for the immobilization of biomolecules, such as proteins or antibodies, in various laboratory applications. It provides a stable environment and facilitates the binding of these molecules to solid supports.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin conjugated horseradish peroxidase, by GE Healthcare (1 mentions)
96 well microtiter plate, by Thermo Fisher Scientific (1 mentions)
Spectrafluor microplate reader, by Tecan (1 mentions)
Abts solution, by Roche (1 mentions)
Abts peroxidase substrate system, by LGC (1 mentions)
Emax plus microplate reader, by Molecular Devices (1 mentions)
2 protocols using coating buffer
1
Recombinant Protein Binding Assay
2
Recombinant PEDV S Protein ELISA
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The recombinant S proteins were diluted to 2 μg/mL with coating buffer (KPL, SeraCare, MA, USA) and were coated on a Nunc maxi-soap plate (Thermo Fisher Scientific) at 4 °C for 16 h. After washing six times with washing buffer (KPL, SeraCare), each well was blocked with 300 μL blocking buffer (KPL, SeraCare) at room temperature for 1 h. The serum samples were diluted 40-fold in blocking buffer and then 100 μL was added per well to washed plates and incubated for 1 h at room temperature. After washing six times, as previously described, HRP-conjugated goat-anti-swine IgG antibody (KPL, SeraCare) was added to the wells at 1:1000 dilution and the plates were then incubated for 1 h at room temperature. The coloration procedure was initiated by applying 50 μL/well of ABTS® Peroxidase Substrate System (KPL, SeraCare) onto the washed plates, and the coloration step was stopped by adding 50 μL/well stopping solution (KPL, SeraCare). The signals were read at 405 nm using the EMax Plus Microplate Reader (Molecular Devices). In the ELISA assays, a double-positive sample for both immunostaining against PEDV-infected Vero cells and commercial PEDV ELISA was chosen as the inter-experimental positive control; similarly, a double-negative serum was chosen as the negative control in this study.
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