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S trimer

Manufactured by ACROBiosystems

The S trimer is a lab equipment product provided by ACROBiosystems. It is a recombinant protein that represents the trimeric spike (S) protein of the SARS-CoV-2 virus. The core function of the S trimer is to serve as a research tool for studying the structure and interactions of the SARS-CoV-2 spike protein.

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4 protocols using s trimer

1

Spike Protein Binding Kinetics

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The simultaneous binding of four antibodies onto the spike protein was tested using a BIAcore 8K system (Cytiva) together with CM5 sensor chips (Cytiva). S trimer (AcroBiosystems) was diluted in pH 5.0 Acetate Buffer (Cytiva) and covalently immobilized on chips using an Amine Coupling Kit (Cytiva). After reaching a ∼ 270 RU coupling level (correspond to ​∼ ​100 RU level of Rmax), the excess antigens were washed away and the unbound sites were blocked with ethanolamine. Antibodies were diluted with HBS-EP buffer (Cytiva) to the concentration of 40 ​μg/mL. The first antibody was injected at a rate of 20 ​μL/min for 600 ​s to reach a saturated binding level, followed by injecting a second antibody into the system. The ascending RU value was monitored to detect the successive binding of four antibodies.
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2

Evaluating ERα Activity in MCF-7 Cells

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The ERα activity was determined by the ERα transcription factor activation assay kit (Abcam, ab207203) according to the manufacturer’s directions. Briefly, MCF-7 nuclear extracts (5 μg; Abcam, ab14860) were treated with S (0.01 to 300 nM; ACROBiosystems), S-RBD (1 to 100 nM; ACROBiosystems), S-trimer (1 to 100 nM; provided by M. Borgnia), and/or E2 (100 nM: Tocris). Extracts were added to each well coated with the ER consensus binding site (5′-GGTCACAGTGACC-3′). The wells were washed and then incubated with rabbit anti-ERα (1:2000; 1 hour at RT) and horseradish peroxidase (HRP)–conjugated secondary antibody (1:2000; 1 hour at RT) that were provided with the kit. Colorimetric reaction was measured by spectrophotometry at a wavelength of 450 nm. Values were given as percentage of the effect induced by E2.
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3

SARS-CoV-2 Antigen-Antibody Binding Assay

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For experiments involving the competitive binding of antibodies to SARS-CoV-2 RBD or S trimer, recombinant hACE2-Fc protein was first biotinylated using EZ-Link Sulfo-NHS-Biotin (ThermoFisher) as the instruction described. SARS-CoV-2 RBD (Sino Biological), S trimer (AcroBiosystems), mutated RBDs (Sino Biological), and mutated S trimers (AcroBiosystems) were coated onto High Binding ELISA 96-Well Plate (BEAVER). In order to obtain an optimized hACE2-Fc concentration for this experiment, the concentration-dependent binding of biotinylated hACE2-Fc to coated SARS-CoV-2 antigens was measured by performing a conventional receptor-binding ELISA. The 80% maximal effective concentration (EC80) of biotinylated hACE2-Fc was calculated by the four-parameter nonlinear fitting. Antibodies were serially diluted in 1% BSA (Sigma) and added 50 μL into the antigen coated plates. Biotinylated hACE2-Fc at EC80 concentration was subsequently pipetted into. After incubation at 37 °C for 1 h, plates were washed four times with PBST and incubated with 100 μL of 1:2000 diluted Ultrasensitive Streptavidin-Peroxidase Polymer (Sigma). After further washing, 100 μL TMB was added, followed by detection of the bound hACE2 in the microplate reader. Four-parameter nonlinear regression fitting in GraphPad Prism Version 9.0.0 was applied for result analysis.
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4

Assay of ERα Transcriptional Activity

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The ERα activity was determined by the ERα transcription factor activation assay kit (ab207203, Abcam) according to manufacturer’s directions. Briefly, MCF-7 nuclear extracts (5 μg; ab14860, Abcam) were treated with either S (0.01–300 nM; Acro Biosystems) S-RBD (1–100 nM; Acro Biosystems), S-trimer (1–100 nM; provided by Dr. Borgnia) and/or E2 (100 nM: Tocris). Extracts were added to each well coated with the ER consensus binding site (5’ – GGTCACAGTGACC – 3’). The wells were washed and then incubated with rabbit anti-ERα (1:2000, 1 h, RT) and horseradish-conjugated secondary antibody (1:2000, 1 h, RT) that were provided with the kit. Colorimetric reaction was measured by spectrophotometry at a wavelength of 450 nm.
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