This assay is based on a split-GFP system (pQCXIP-GFP1-10 and pQCXIP-GFP11) where GFP signals are produced upon cell fusion (Kodaka et al., 2015 (link)). The split GFP plasmids: pQCXIP-BSR-GFP1-10 and pQCXIP-GFP11 were a gift from Yutaka Hata (Addgene plasmid #68715, https://www.addgene.org/68715/ and #68716 https://www.addgene.org/68716/ ). Briefly, 200,000 Vero-TMPRSS2 cells were co-transfected with 500 ng pCAGGS- spike and 500 ng pQCXIP-GFP1-10 or pQCXIP-GFP11 using TransIT-LT1 (Takara; MIR2300). The next day, cells were detached, pooled and reseeded in black 96-well or 24-well plates (Greiner, M0562-32EA) at 25,000 and 200,000 total cells/well, respectively. After 24 h, cells were fixed with 4% paraformaldehyde in PBS for 10 min. To measure spike expression, cells were permeabilized with 0.1% Triton X-100 for 15 min and stained with primary antibody anti-S2 (Sino Biological, 40590-D001) and secondary Alexa Fluor 594 goat anti-human antibody (Thermo Fisher, A-11014). DAPI was used to visualize the nucleus. Images of the whole wells were obtained and analyzed using the Celigo Image Cytometer (Nexcelom). GFP signals were normalized to spike expression and DAPI counts to represent fusion activity.
Anti s2
Manufactured by Sino Biological
Anti-S2 is a recombinant protein that corresponds to the S2 subunit of the SARS-CoV-2 spike protein. It can be used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg hrp, by Merck Group (5 mentions)
Immobilon crescendo western hrp substrate, by Merck Group (4 mentions)
Amersham imager 600, by GE Healthcare (4 mentions)
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
Protease inhibitor, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Anti mouse igg peroxidase, by Merck Group (2 mentions)
Anti s1, by Sino Biological (2 mentions)
Anti sars cov 2 s1, by Sino Biological (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Transit lt1, by Takara Bio (1 mentions)
M0562 32ea, by Greiner (1 mentions)
Alexa fluor 594 goat anti human antibody, by Thermo Fisher Scientific (1 mentions)
Celigo image cytometer, by Revvity (1 mentions)
Anit gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti mouse igg fitc, by Merck Group (1 mentions)
Imager 600, by GE Healthcare (1 mentions)
Immobolin crescendo western hrp substrate, by Merck Group (1 mentions)
Anti rabbit igg fitc, by Merck Group (1 mentions)
Sc 47724, by Santa Cruz Biotechnology (1 mentions)
8 protocols using anti s2
1
Split-GFP Assay for Measuring Cell Fusion
2
SARS-CoV-2 Spike Protein Detection
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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.
3
Quantifying SARS-CoV-2 Spike Subunit Ratios
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HEK293T cells producing pseudotyped virus were used to determine the extent to which the spikes were being processed into S1/S2 subunits. Lysates were collected at 72 h post-transfection through a RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, Nonidet P-40, 0.1% SDS) lysis supplemented with protease inhibitor cocktails (Sigma, P8340). The lysis was performed for 40 min and subjected to SDS-PAGE and western blotting. Blots were probed with anti-S2 (Sino Biological, 40590; RRI-D:AB_2857932), anti-p24 (NIH HIV Reagent Program, ARP-1513), and anti-GAPDH (Santa Cruz, Cat# sc-47724, RRID: AB_627678). Secondary antibodies used for blotting included Anti-Rabbit-IgG-HRP (Sigma, A9169; RRID:AB_258434) and Anti-Mouse (Sigma, Cat# A5278, RRID: AB_258232). Blots were performed using Immobilon Crescendo Western HRP substrate (Millipore, WBLUR0500) and exposed on a GE Amersham Imager 600. Quantification of band intensity was performed with NIH ImageJ (Bethesda, MD). ImageJ quantification was used to calculate an S2/S ratio that was normalized to D614G (D614G = 1.0).
4
Quantifying SARS-CoV-2 Spike Protein Expression
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HEK293T cells transfected with spike of interest were lysed in 300 µL RIPA + PI + PMSF (RIPA: 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, Nonidet P-40, 0.1% SDS, Protease inhibitor cocktail: Sigma, P8340) for 40 min on ice. Lysate was harvested and used for western blotting. Samples were run on a 10% acrylamide SDS-PAGE gel and transferred to a PVDF membrane. Blots were probed with anti-S2 (Sino Biological, 40590; RRID:AB_2857932) and anti-GAPDH as a loading control (Santa Cruz, Cat# sc-47724, RRID: AB_627678). Secondary antibodies included anti-Rabbit-IgG-FITC (Sigma, A9169; RRID:AB_258434) and anti-Mouse-IgG-FITC (Sigma, Cat# A5278, RRID: AB_258232). Blots were imaged using Immobolin Crescendo Western HRP substrate (Millipore, WBLUR0500) and exposed on a GE Amersham Imager 600. Quantification of band intensity was determined using ImageJ (NIH, Bethesda, MD).
5
SARS-CoV-2 Spike Protein Cleavage Assay
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Calu-3 cells were pre-treated by camostat (Cam, 50 μg ml−1), 8P9R (50 μg ml−1) or PBS for 1 h and then S protein (1 μg) of SARS-CoV-2 was added to treated cells for 1 h to let TMPRSS2 to cleave S protein. S protein without cells and drugs was served as the no digestion control. Samples were lysed in 1× Passive Lysis Buffer (Promega, E194A) and applied to detect spike protein using anti-S2 (Sino biological, 40590-T62, 1:5000 dilution) and anti-rabbit HRP as secondary antibody (Thermo Scientific, 31460, 1:4000 dilution).
6
Quantitative Analysis of SARS-CoV-2 Viral Proteins
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The remaining 293T cells used to produce lentiviral vectors are lysed in RIPA buffer (Sigma-Aldrich, R0278) supplemented with protease inhibitor (Sigma, P8340) for 40 minutes on ice. Lysate is collected and a portion is used for SDS-PAGE on a 10% poly-acrylamide gel and transferred to a PVDF membrane for western blotting. Blots were probed with polyclonal anti-SARS-CoV-2 S1 (Sino Biological, 40591-T62; RRID:AB_2893171), anti-S2 (Sino Biological, 40590; RRID:AB_2857932), anti-p24 (NIH HIV Reagent Program, ARP-1513), and anti-GAPDH (Santa Cruz, Cat# sc-47724, RRID: AB_627678). Secondary antibodies used were Anti-Rabbit-IgG-HRP (Sigma, A9169; RRID:AB_258434) and Anti-Mouse (Sigma, Cat# A5278, RRID: AB_258232). Blots were visualized via Immobilon Crescendo Western HRP substrate (Millipore, WBLUR0500) and exposed on a GE Amersham Imager 600. Band intensities were quantified using NIH Image J analysis software (Bethesda, MD).
7
Characterization of SARS-CoV-2 Viral Proteins
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The remaining 293T cells used to produce lentiviral vectors are lysed in RIPA buffer (Sigma-Aldrich, R0278) supplemented with protease inhibitor (Sigma, P8340) for 40 min on ice. Lysate is collected and a portion is used for SDS-PAGE on a 10% poly-acrylamide gel and transferred to a PVDF membrane for western blotting. Blots were probed with polyclonal anti-SARS-CoV-2 S1 (Sino Biological, 40591-T62; RRID:AB_2893171), anti-S2 (Sino Biological, 40590; RRID:AB_2857932), anti-p24 (NIH HIV Reagent Program, ARP-1513), and anti-GAPDH (Santa Cruz, Cat# sc-47724, RRID: AB_627678). Secondary antibodies used were anti-Rabbit-IgG-HRP (Sigma, A9169; RRID:AB_258434) and anti-Mouse (Sigma, Cat# A5278, RRID: AB_258232). Blots were visualized via Immobilon Crescendo Western HRP substrate (Millipore, WBLUR0500) and exposed on a GE Amersham Imager 600. Band intensities were quantified using NIH Image J analysis software (Bethesda, MD).
8
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.
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