Western blotting was conducted as previously described (Zeng et al., 2020 (link)). Briefly, cells were collected and lysed in 200 ul of RIPA buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, Nonidet P-40, 0.1% SDS) in the presence of protease inhibitor cocktail (MilliporeSigma, P8340), followed by clarification at 13200 rpm for 10 minutes, and boiling for 10 minutes at 100°C with 1x SDS loading buffer. To determine the Spike content in virion particles, pseudovirus supernatant was collected, filtered and purified by ultracentrifugation through a 20% sucrose cushion. The purified virions were dissolved in 1x SDS loading buffer. Subsequently, the samples were separated by 10% SDS-PAGE gels, transferred to PVDF membranes and immunoblotted with anti-S1 (Sino Biological, 40150-T62), anti-S2 (Sino Biological, 40590-T62), anit-GAPDH (Santa Cruz, sc-47724), and anti-p24 (anti-p24 (NIH ARP-1513) antibodies, followed by immunoblotting with anti-mouse-IgG-Peroxidase (Sigma, A5278) or anti-rabbit-IgG-HRP (Sigma, A9169) antibodies.
Anti s1
Manufactured by Sino Biological
Sourced in China
Anti-S1 is a laboratory reagent produced by Sino Biological. It is an antibody that specifically binds to the S1 subunit of the SARS-CoV-2 spike protein. The core function of Anti-S1 is to serve as a tool for research and development related to SARS-CoV-2 and COVID-19.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse igg peroxidase, by Merck Group (4 mentions)
Anti rabbit igg hrp, by Merck Group (4 mentions)
Anit gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti s2, by Sino Biological (2 mentions)
Immobilon crescendo western hrp substrate, by Merck Group (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Amersham imager, by Cytiva (2 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Sars cov 2 nucleocapsid protein, by Sino Biological (1 mentions)
Anti β actin, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti p24, by Abcam (1 mentions)
Anti flag beads, by Merck Group (1 mentions)
5 protocols using anti s1
1
SARS-CoV-2 Spike Protein Detection
2
SARS-CoV-2 Antibody Detection ELISA Assay
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In this study, two in-house enzyme-linked immunosorbent assay (ELISA) systems were used to assess the participants’ anti-spike IgG (anti-S1 + RBD IgG) and anti-N IgG antibody levels [17 (link),18 (link),19 (link)]. Briefly, ELISA plates were pre-coated with SARS-CoV-2 Spike 1 and RBD proteins (Sino biological, Beijing, China) at a ratio of 6:4, and SARS-CoV-2 nucleocapsid protein (Sino biological, Beijing, China) for anti-spike IgG (anti-S1 + RBD IgG) and anti-N IgG detection. After the serum separation, the serum was diluted (1:100) with dilution buffer and dispensed into wells with positive, negative, and plate controls. After incubation for 15 min and a washing step, horseradish peroxidase-conjugated anti-human IgG (The NativeAntigens, London, UK) at a 1:4000 dilution was added to the wells. After a short incubation and wash, the substrate, 3,3′,5,5′-Tetramethylbenzidine (TMB), was added to each well, followed by a stop solution. The optical density of the final reaction was measured at 450 nm. The antibody level was finally determined by analyzing the OD/cut-off ratio.
3
SARS-CoV-2 Spike Protein Analysis
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Lysate was collected from virus producer cells through a 30-minute incubation on ice in RIPA lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, Nonidet P-40, 0.1% SDS) supplemented with protease inhibitor (Sigma, P8340). Samples were run on a 10% acrylamide SDS-PAGE gel and transferred to a PVDF membrane. Membranes were probed with anti-S1 (Sino Biological, 40591-T62; RRID:AB_2893171), anti-S2 (Sino Biological, 40590; RRID:AB_2857932), and anti-β-actin (ThermoFisher, MA5–15740; RRID:AB_10983927). Secondary antibodies included Anti-mouse-IgG-Peroxidase (Sigma, A5278; RRID:AB_258232) and Anti-rabbit-IgG-HRP (Sigma, A9169; RRID:AB_258434). Blots were imaged using Immobilon Crescendo Western HRP substrate (Millipore, WBLUR0500) on a GE Amersham Imager 600. Band intensities were quantified using NIH ImageJ (Bethesda, MD) image analysis software.
4
Purification and Western Blot of Pseudotyped Viruses
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Pseudotyped virus particles were purified by ultracentrifugation through a 20% sucrose cushion. Virus was resuspended in SDS-PAGE loading buffer. Cell lysate from virus producer cells was collected by 30 min incubation of cells on ice in RIPA lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, Nonidet P-40, 0.1% SDS) supplemented with protease inhibitor (Sigma, P8340). Samples were run on a 10% acrylamide SDS-PAGE gel and transferred to a PVDF membrane. Membranes were probed with anti-Flag (Sigma, F3165), anti-p24 (Abcam, ab63917; NIH ARP-1513), anti-S1 (Sino Biological, 40150-T62), and anit-β-actin (Sigma, A1978). Anti-mouse-IgG-Peroxidase (Sigma, A5278) and anti-rabbit-IgG-HRP (Sigma, A9169) were used as secondary antibodies where appropriate. Blots were imaged with Immobilon Crescendo Western HRP substrate (Millipore, WBLUR0500) on a GE Amersham Imager 600.
5
SARS-CoV-2 S-protein Shedding Assay
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HEK293T cells were transfected with S expression constructs. Then, 24 hrs after transfection, cells were treated with or without sACE2 (25 μg/mL) for 4 hrs at 37°C. Cell lysate and cell culture media was harvested. S1-containig cell culture media was incubated with 10 μL of anti-Flag beads (Sigma, F2426) to precipitate S1 subunit. Following immune-precipitation, cell lysate and shed S1 were run on 10% SDS-PAGE gel, transferred, and probed with anti-S1 (Sino Biological, 40150-T62) and anit-GAPDH (Santa Cruz, sc-47724). Anti-mouse-IgG-Peroxidase (Sigma, A5278) and anti-rabbit-IgG-HRP (Sigma, A9169) were used as secondary antibodies.
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