anti flag antibody produced in rabbit  (Sino Biological)


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    Name:
    ACE2 Angiotensin Converting Enzyme 2 Antibody Rabbit MAb
    Description:
    This antibody was obtained from a rabbit immunized with purified recombinant Rat ACE2 rR ACE2 Catalog 80031 R08H Q5EGZ1 Met1 Thr740
    Catalog Number:
    80031-r039
    Price:
    None
    Applications:
    IHC-P
    Host:
    Rabbit
    Immunogen:
    Recombinant Rat ACE2 protein (Catalog#80031-R08H)
    Category:
    Primary Antibody
    Antibody Type:
    MAb
    Isotype:
    Rabbit IgG
    Reactivity:
    Rat
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    Structured Review

    Sino Biological anti flag antibody produced in rabbit
    Identification of host factors associated with N protein and phosphorylation on N protein by mass spectrometry. (A) <t>Flag</t> tagged N protein was immunoprecipitated from Caco-2-N cells infected with recombinant SARS-CoV-2 GFP/ΔN trVLP using Flag antibody, and the proteins were analyzed on SDS-PAGE gel. The proteins were visualized by Coomassie blue staining. N, G3BP1 and G3BP2 were labelled. (B) Phosphorylated peptides of N protein derived from Caco-2-N cells. Caco-2-N cells in which N was C-terminal Flag-tagged were collected and cell lysates were immunoprecipitated with <t>anti-Flag</t> coupled beads. Phosphorylated peptides of the immunoprecipitates were analyzed by mass spectrometry.
    This antibody was obtained from a rabbit immunized with purified recombinant Rat ACE2 rR ACE2 Catalog 80031 R08H Q5EGZ1 Met1 Thr740
    https://www.bioz.com/result/anti flag antibody produced in rabbit/product/Sino Biological
    Average 93 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    anti flag antibody produced in rabbit - by Bioz Stars, 2021-02
    93/100 stars

    Images

    1) Product Images from "A novel cell culture system modeling the SARS-CoV-2 life cycle"

    Article Title: A novel cell culture system modeling the SARS-CoV-2 life cycle

    Journal: bioRxiv

    doi: 10.1101/2020.12.13.422469

    Identification of host factors associated with N protein and phosphorylation on N protein by mass spectrometry. (A) Flag tagged N protein was immunoprecipitated from Caco-2-N cells infected with recombinant SARS-CoV-2 GFP/ΔN trVLP using Flag antibody, and the proteins were analyzed on SDS-PAGE gel. The proteins were visualized by Coomassie blue staining. N, G3BP1 and G3BP2 were labelled. (B) Phosphorylated peptides of N protein derived from Caco-2-N cells. Caco-2-N cells in which N was C-terminal Flag-tagged were collected and cell lysates were immunoprecipitated with anti-Flag coupled beads. Phosphorylated peptides of the immunoprecipitates were analyzed by mass spectrometry.
    Figure Legend Snippet: Identification of host factors associated with N protein and phosphorylation on N protein by mass spectrometry. (A) Flag tagged N protein was immunoprecipitated from Caco-2-N cells infected with recombinant SARS-CoV-2 GFP/ΔN trVLP using Flag antibody, and the proteins were analyzed on SDS-PAGE gel. The proteins were visualized by Coomassie blue staining. N, G3BP1 and G3BP2 were labelled. (B) Phosphorylated peptides of N protein derived from Caco-2-N cells. Caco-2-N cells in which N was C-terminal Flag-tagged were collected and cell lysates were immunoprecipitated with anti-Flag coupled beads. Phosphorylated peptides of the immunoprecipitates were analyzed by mass spectrometry.

    Techniques Used: Mass Spectrometry, Immunoprecipitation, Infection, Recombinant, SDS Page, Staining, Derivative Assay

    2) Product Images from "Difference in levels of SARS-CoV-2 S1 and S2 subunits- and nucleocapsid protein-reactive SIgM/IgM, IgG and SIgA/IgA antibodies in human milk"

    Article Title: Difference in levels of SARS-CoV-2 S1 and S2 subunits- and nucleocapsid protein-reactive SIgM/IgM, IgG and SIgA/IgA antibodies in human milk

    Journal: Journal of Perinatology

    doi: 10.1038/s41372-020-00805-w

    The levels of SARS-CoV-2-reactive to S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic. The levels of a S1 + S2- and b nucleocapsid-reactive secretory IgM (SIgM)/IgM, IgG, and secretory IgA (SIgA)/IgA in human milk. Values are mean ± SD, n = 41 for women. Asterisks show statistically significant differences between variables (*** p
    Figure Legend Snippet: The levels of SARS-CoV-2-reactive to S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic. The levels of a S1 + S2- and b nucleocapsid-reactive secretory IgM (SIgM)/IgM, IgG, and secretory IgA (SIgA)/IgA in human milk. Values are mean ± SD, n = 41 for women. Asterisks show statistically significant differences between variables (*** p

    Techniques Used:

    Regression linear between antibodies reactive to SARS-CoV-2 S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic in 41 women. a Positive correlation of S1 + S2-reactive secretory IgA (SIgA)/IgA and S1 + S2-reactive secretory IgM (SIgM)/IgM. b Positive correlation between S1 + S2-reactive SIgA/IgA and nucleocapsid-reactive SIgA/IgA. c Positive correlation between nucleocapsid-reactive SIgA/IgA and SIgM/IgM. d Positive correlation between nucleocapsid-reactive SIgM/IgM and S1 + S2-reactive SIgM/IgM. Pearson correlation coefficients ( r ) were determined when p
    Figure Legend Snippet: Regression linear between antibodies reactive to SARS-CoV-2 S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic in 41 women. a Positive correlation of S1 + S2-reactive secretory IgA (SIgA)/IgA and S1 + S2-reactive secretory IgM (SIgM)/IgM. b Positive correlation between S1 + S2-reactive SIgA/IgA and nucleocapsid-reactive SIgA/IgA. c Positive correlation between nucleocapsid-reactive SIgA/IgA and SIgM/IgM. d Positive correlation between nucleocapsid-reactive SIgM/IgM and S1 + S2-reactive SIgM/IgM. Pearson correlation coefficients ( r ) were determined when p

    Techniques Used:

    The levels of antibodies reactive to SARS-CoV-2 S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic (2020-HM) and 2 years prior this pandemic (2018-HM). Levels of S1 + S2-reactive a secretory IgM (SIgM)/IgM, b IgG and c secretory IgA (SIgA)/IgA in 2020-HM and 2018-HM. Levels of nucleocapsid-reactive d SIgM/IgM, e IgG and f SIgA/IgA in 2020-HM and 2018-HM. Values are mean ± SD, n = 41 for 2020-HM and n = 16 for 2018-HM. Asterisks show statistically significant differences between variables (* p
    Figure Legend Snippet: The levels of antibodies reactive to SARS-CoV-2 S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic (2020-HM) and 2 years prior this pandemic (2018-HM). Levels of S1 + S2-reactive a secretory IgM (SIgM)/IgM, b IgG and c secretory IgA (SIgA)/IgA in 2020-HM and 2018-HM. Levels of nucleocapsid-reactive d SIgM/IgM, e IgG and f SIgA/IgA in 2020-HM and 2018-HM. Values are mean ± SD, n = 41 for 2020-HM and n = 16 for 2018-HM. Asterisks show statistically significant differences between variables (* p

    Techniques Used:

    Percentage of detected antibodies reactive to S1 and S2 subunits (S1 + S2), and nucleocapsid from SARS-CoV-2 in human milk collected during the COVID-19 pandemic. S1+S2-reactive a secretory IgM (SIgM)/IgM, b IgG and c secretory IgA (SIgA)/IgA in human milk from 41 women. Nucleocapsid-reactive d SIgM/IgM, e IgG and f SIgA/IgA. Milk expression period was from 30/02/20 to 03/04/20 in the United States.
    Figure Legend Snippet: Percentage of detected antibodies reactive to S1 and S2 subunits (S1 + S2), and nucleocapsid from SARS-CoV-2 in human milk collected during the COVID-19 pandemic. S1+S2-reactive a secretory IgM (SIgM)/IgM, b IgG and c secretory IgA (SIgA)/IgA in human milk from 41 women. Nucleocapsid-reactive d SIgM/IgM, e IgG and f SIgA/IgA. Milk expression period was from 30/02/20 to 03/04/20 in the United States.

    Techniques Used: Expressing

    3) Product Images from "3-Hydroxyphthalic Anhydride-Modified Chicken Ovalbumin as a Potential Candidate Inhibits SARS-CoV-2 Infection by Disrupting the Interaction of Spike Protein With Host ACE2 Receptor"

    Article Title: 3-Hydroxyphthalic Anhydride-Modified Chicken Ovalbumin as a Potential Candidate Inhibits SARS-CoV-2 Infection by Disrupting the Interaction of Spike Protein With Host ACE2 Receptor

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2020.603830

    Inhibitory activity of HP-OVA against SARS-CoV-2 S-mediated cell-cell fusion. Images were captured at 12 h after treatment with HP-OVA or OVA on SARS-CoV-2 S protein-mediated cell-cell fusion. The syncytia of Vero E6 cells (A) or Huh 7 cells (B) and HEK293T cells with SARS-CoV-2 overexpression are marked in the pictures. Representative results from three fields were selected randomly from each sample with scale bars of 50 μm (C, D) The number of syncytia was counted under an inverted fluorescence microscope, and the percentage of inhibition was calculated as described in the Methods. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p
    Figure Legend Snippet: Inhibitory activity of HP-OVA against SARS-CoV-2 S-mediated cell-cell fusion. Images were captured at 12 h after treatment with HP-OVA or OVA on SARS-CoV-2 S protein-mediated cell-cell fusion. The syncytia of Vero E6 cells (A) or Huh 7 cells (B) and HEK293T cells with SARS-CoV-2 overexpression are marked in the pictures. Representative results from three fields were selected randomly from each sample with scale bars of 50 μm (C, D) The number of syncytia was counted under an inverted fluorescence microscope, and the percentage of inhibition was calculated as described in the Methods. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Techniques Used: Activity Assay, Over Expression, Fluorescence, Microscopy, Inhibition

    Inhibition of HP-OVA on the infection with SARS-CoV-2 PsV and SARS-CoV PsV. Antiviral activity of HP-OVA against SARS-CoV-2 S PsV infection in 293T/ACE2 (A) or Vero E6 (B) target cells. Inhibition of single-round infection of SARS-CoV S PsV in 293T/ACE2 (C) and Vero E6 (D) cells. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p
    Figure Legend Snippet: Inhibition of HP-OVA on the infection with SARS-CoV-2 PsV and SARS-CoV PsV. Antiviral activity of HP-OVA against SARS-CoV-2 S PsV infection in 293T/ACE2 (A) or Vero E6 (B) target cells. Inhibition of single-round infection of SARS-CoV S PsV in 293T/ACE2 (C) and Vero E6 (D) cells. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Techniques Used: Inhibition, Infection, Activity Assay

    HP-OVA binding to both SARS-CoV-2 S and ACE2 protein. Analysis of the expression of SARS-CoV-2 S (A) and ACE2 (B) in HEK-293T cells by western blot. The binding of HP-OVA to cells expressing SARS-CoV-2 S (C) or ACE2 (D) was assessed by flow cytometry. A representative flow histogram and quantification of the binding of HP-OVA to cells expressing SARS-CoV-2 S (E) or ACE2 (F) were shown. Data are presented as the mean ± SD (* p
    Figure Legend Snippet: HP-OVA binding to both SARS-CoV-2 S and ACE2 protein. Analysis of the expression of SARS-CoV-2 S (A) and ACE2 (B) in HEK-293T cells by western blot. The binding of HP-OVA to cells expressing SARS-CoV-2 S (C) or ACE2 (D) was assessed by flow cytometry. A representative flow histogram and quantification of the binding of HP-OVA to cells expressing SARS-CoV-2 S (E) or ACE2 (F) were shown. Data are presented as the mean ± SD (* p

    Techniques Used: Binding Assay, Expressing, Western Blot, Flow Cytometry

    The interaction of HP-OVA with SARS-CoV-2 S and ACE2. The binding of OVA to SARS-CoV-2 spike (RBD), S2 and BSA protein was assessed by ELISA (A) . The binding of HP-OVA to SARS-CoV-2 spike (RBD), S2 and a negative control BSA protein was assessed by ELISA (B) . The binding ability of HP-OVA to ACE2 protein was assessed by ELISA (C) . Inhibition of the interaction between spike (RBD) and ACE2 proteins by HP-OVA, as determined by a competitive inhibition ELISA (D) . Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p
    Figure Legend Snippet: The interaction of HP-OVA with SARS-CoV-2 S and ACE2. The binding of OVA to SARS-CoV-2 spike (RBD), S2 and BSA protein was assessed by ELISA (A) . The binding of HP-OVA to SARS-CoV-2 spike (RBD), S2 and a negative control BSA protein was assessed by ELISA (B) . The binding ability of HP-OVA to ACE2 protein was assessed by ELISA (C) . Inhibition of the interaction between spike (RBD) and ACE2 proteins by HP-OVA, as determined by a competitive inhibition ELISA (D) . Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Inhibition

    Schematic representation of the molecular mechanisms of HP-OVA against SARS-CoV-2 infection. HP-OVA binds to both the S protein of SARS-CoV-2 and host angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV-2, and disrupts the S protein-ACE2 interaction, thereby exhibiting inhibitory activity against SARS-CoV-2 infection.
    Figure Legend Snippet: Schematic representation of the molecular mechanisms of HP-OVA against SARS-CoV-2 infection. HP-OVA binds to both the S protein of SARS-CoV-2 and host angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV-2, and disrupts the S protein-ACE2 interaction, thereby exhibiting inhibitory activity against SARS-CoV-2 infection.

    Techniques Used: Infection, Functional Assay, Activity Assay

    4) Product Images from "Distinct conformational states of SARS-CoV-2 spike protein"

    Article Title: Distinct conformational states of SARS-CoV-2 spike protein

    Journal: bioRxiv

    doi: 10.1101/2020.05.16.099317

    A model for structural rearrangements of SARS-Cov-2 S protein. (A) Structural changes independent of a target cell. We suggest that both the prefusion and postfusion spikes are present on the surface of mature virion and the ratio between them may vary (diagram of virion). The postfusion spikes on the virion are formed by S2 after S1 dissociates in the absence of ACE2. (B) ACE2-dependent structural rearrangements. Structural transition from the prefusion to postfusion conformation inducing membrane fusion likely proceeds stepwise as follows: 1) FPPR clamps down RBD through CTD1 in the prefusion S trimer, but it occasionally flips out of position and allows an RBD to sample the up conformation. 2) RBD binding to ACE2 creates a flexible FPPR that enables exposure of the S2’ cleavage site immediately upstream of the adjacent fusion peptide (FP). Cleavage at the S2’ site, and perhaps also the S1/S2 site, releases the structural constraints on the fusion peptide and initiates a cascade of refolding events in S2, probably accompanied by complete dissociation of S1. 3) Formation of the long central three-stranded coiled-coil and folding back of HR2. 4) Formation of the postfusion structure of S2 that brings the two membranes together, facilitating formation of a fusion pore and viral entry.
    Figure Legend Snippet: A model for structural rearrangements of SARS-Cov-2 S protein. (A) Structural changes independent of a target cell. We suggest that both the prefusion and postfusion spikes are present on the surface of mature virion and the ratio between them may vary (diagram of virion). The postfusion spikes on the virion are formed by S2 after S1 dissociates in the absence of ACE2. (B) ACE2-dependent structural rearrangements. Structural transition from the prefusion to postfusion conformation inducing membrane fusion likely proceeds stepwise as follows: 1) FPPR clamps down RBD through CTD1 in the prefusion S trimer, but it occasionally flips out of position and allows an RBD to sample the up conformation. 2) RBD binding to ACE2 creates a flexible FPPR that enables exposure of the S2’ cleavage site immediately upstream of the adjacent fusion peptide (FP). Cleavage at the S2’ site, and perhaps also the S1/S2 site, releases the structural constraints on the fusion peptide and initiates a cascade of refolding events in S2, probably accompanied by complete dissociation of S1. 3) Formation of the long central three-stranded coiled-coil and folding back of HR2. 4) Formation of the postfusion structure of S2 that brings the two membranes together, facilitating formation of a fusion pore and viral entry.

    Techniques Used: Binding Assay

    Cryo-EM structure of the SARS-CoV-2 S2 in the postfusion conformation. (A) The structure of the S2 trimer was modeled based on a 3.3Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of the S2 trimer in the postfusion conformation shown in ribbon diagram. Various structural components in the color scheme shown in Fig. 1A include HR1, heptad repeat 1; CH, central helix region; CD, connector domain; and HR1, heptad repeat 2. The S2’ cleavage site is in a disordered loop between Ile770 and Thr912. A possible location of the S2 N-terminus (S1/S2 cleavage site) is also indicated. (C) A low resolution map showing the density pattern for 5 N-linked glycans, with almost equal spacing along the long axis.
    Figure Legend Snippet: Cryo-EM structure of the SARS-CoV-2 S2 in the postfusion conformation. (A) The structure of the S2 trimer was modeled based on a 3.3Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of the S2 trimer in the postfusion conformation shown in ribbon diagram. Various structural components in the color scheme shown in Fig. 1A include HR1, heptad repeat 1; CH, central helix region; CD, connector domain; and HR1, heptad repeat 2. The S2’ cleavage site is in a disordered loop between Ile770 and Thr912. A possible location of the S2 N-terminus (S1/S2 cleavage site) is also indicated. (C) A low resolution map showing the density pattern for 5 N-linked glycans, with almost equal spacing along the long axis.

    Techniques Used:

    Selected new features of the SARS-CoV-2 prefusion S trimer. (A) N-terminal segment of S protein. The N-terminus is at residue Gln14 after cleavage of the signal peptide. Cys15 forms a disulfide bond with Cys136. We observed good density for the N-linked glycan at Asn17. (B) A segment immediately downstream of the fusion peptide, while disordered in the stabilized soluble S ectodomain trimer structure, forms a tightly packed structure, designated FPPR for the fusion peptide proximal region, abutting CTD1. The newly identified FPPR structure would clash with CTD1 in the RBD up conformation. Various domains are shown in the color scheme in Fig. 2B . The structure of the soluble S trimer with one RBD in the up conformation (PDB ID: 6vyb) is shown in gray. In the box, a close-up view of the FPPR with adjacent fusion peptide in both surface representation and stick model. (C) The SARS-CoV-2 prefusion S trimer, viewed along the threefold axis, is superposed on the structure of the stabilized soluble S ectodomain trimer in the closed conformation with all three RBDs in the down conformation (PDB ID: 6vxx). While the S2 region is well aligned, there is a significant shift (e.g. ∼10Å between two Ala123 residues) in S1. (D) Impact of the proline mutations introduced at residues 986 and 987 to stabilize the prefusion conformation. K986P mutation removes a salt bridge between Lys986 of one protomer and either Asp426 or Asp427 of another protomer in the trimer interface.
    Figure Legend Snippet: Selected new features of the SARS-CoV-2 prefusion S trimer. (A) N-terminal segment of S protein. The N-terminus is at residue Gln14 after cleavage of the signal peptide. Cys15 forms a disulfide bond with Cys136. We observed good density for the N-linked glycan at Asn17. (B) A segment immediately downstream of the fusion peptide, while disordered in the stabilized soluble S ectodomain trimer structure, forms a tightly packed structure, designated FPPR for the fusion peptide proximal region, abutting CTD1. The newly identified FPPR structure would clash with CTD1 in the RBD up conformation. Various domains are shown in the color scheme in Fig. 2B . The structure of the soluble S trimer with one RBD in the up conformation (PDB ID: 6vyb) is shown in gray. In the box, a close-up view of the FPPR with adjacent fusion peptide in both surface representation and stick model. (C) The SARS-CoV-2 prefusion S trimer, viewed along the threefold axis, is superposed on the structure of the stabilized soluble S ectodomain trimer in the closed conformation with all three RBDs in the down conformation (PDB ID: 6vxx). While the S2 region is well aligned, there is a significant shift (e.g. ∼10Å between two Ala123 residues) in S1. (D) Impact of the proline mutations introduced at residues 986 and 987 to stabilize the prefusion conformation. K986P mutation removes a salt bridge between Lys986 of one protomer and either Asp426 or Asp427 of another protomer in the trimer interface.

    Techniques Used: Mutagenesis

    Preparation of a full-length SARS-CoV-2 spike protein. (A) Schematic representation of the expression construct of full-length SARS-CoV-2 spike (S) protein. Segments of S1 and S2 include: NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; S1/S2, S1/S2 cleavage site; S2’, S2’ cleavage site; FP, fusion peptide; FFPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane anchor; CT, cytoplasmic tail; and tree-like symbols for glycans. A strep-tag was fused to the C-terminus of S protein by a flexible linker. (B) The purified S protein was resolved by gel-filtration chromatography on a Superose 6 column in the presence of detergent NP-40. The molecular weight standards include thyoglobulin (670 kDa), γ-globulin (158 kDa) and ovalbumin (44 kDa). Three major peaks (peak I-III) contain the S protein. (C) Load sample and peak fractions from (B) were analyzed by Coomassie stained SDS-PAGE. Labeled bands were confirmed by western blot (S, S1 and S2) or protein sequencing (S2 and Cont; S and S1 bands did not gave any meaningful results probably due to a blocked N-terminus). Cont, copurified contaminating protein, identified as endoplasmic reticulum chaperone BiP precursor by N-terminal sequencing. Representative images and 2D averages by negative stain EM of three peak fractions are also shown. The box size of 2D averages is ∼510Å.
    Figure Legend Snippet: Preparation of a full-length SARS-CoV-2 spike protein. (A) Schematic representation of the expression construct of full-length SARS-CoV-2 spike (S) protein. Segments of S1 and S2 include: NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; S1/S2, S1/S2 cleavage site; S2’, S2’ cleavage site; FP, fusion peptide; FFPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane anchor; CT, cytoplasmic tail; and tree-like symbols for glycans. A strep-tag was fused to the C-terminus of S protein by a flexible linker. (B) The purified S protein was resolved by gel-filtration chromatography on a Superose 6 column in the presence of detergent NP-40. The molecular weight standards include thyoglobulin (670 kDa), γ-globulin (158 kDa) and ovalbumin (44 kDa). Three major peaks (peak I-III) contain the S protein. (C) Load sample and peak fractions from (B) were analyzed by Coomassie stained SDS-PAGE. Labeled bands were confirmed by western blot (S, S1 and S2) or protein sequencing (S2 and Cont; S and S1 bands did not gave any meaningful results probably due to a blocked N-terminus). Cont, copurified contaminating protein, identified as endoplasmic reticulum chaperone BiP precursor by N-terminal sequencing. Representative images and 2D averages by negative stain EM of three peak fractions are also shown. The box size of 2D averages is ∼510Å.

    Techniques Used: Expressing, Construct, Binding Assay, Strep-tag, Purification, Filtration, Chromatography, Molecular Weight, Staining, SDS Page, Labeling, Western Blot, Sequencing

    Cryo-EM structure of the SARS-CoV-2 S protein in the prefusion conformation. (A) The structure of the S trimer was modeled based on a 3.1Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of S protein in the prefusion conformation shown in ribbon representation. Various structural components in the color scheme shown in Fig. 1A include NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; and CD, connector domain. N-terminus, S1/S2 cleavage site and S2’ cleavage site are indicated.
    Figure Legend Snippet: Cryo-EM structure of the SARS-CoV-2 S protein in the prefusion conformation. (A) The structure of the S trimer was modeled based on a 3.1Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of S protein in the prefusion conformation shown in ribbon representation. Various structural components in the color scheme shown in Fig. 1A include NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; and CD, connector domain. N-terminus, S1/S2 cleavage site and S2’ cleavage site are indicated.

    Techniques Used: Binding Assay

    5) Product Images from "A novel cell culture system modeling the SARS-CoV-2 life cycle"

    Article Title: A novel cell culture system modeling the SARS-CoV-2 life cycle

    Journal: bioRxiv

    doi: 10.1101/2020.12.13.422469

    Generation of Caco-2 cell expressing SARS-CoV-2 N by lentiviral transduction. (A) Scheme depicting the bicistronic lentiviral constructs for expressing SARS-CoV-2 N protein with C-terminal Flag tag. (B) Representative flow cytometry plots demonstrating efficient lentivirus transduction. Caco-2 cells were transduced with pLVX-N-Flag-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of N-expressing cells. The flow cytometry result was representative of one of three independent experiments.
    Figure Legend Snippet: Generation of Caco-2 cell expressing SARS-CoV-2 N by lentiviral transduction. (A) Scheme depicting the bicistronic lentiviral constructs for expressing SARS-CoV-2 N protein with C-terminal Flag tag. (B) Representative flow cytometry plots demonstrating efficient lentivirus transduction. Caco-2 cells were transduced with pLVX-N-Flag-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of N-expressing cells. The flow cytometry result was representative of one of three independent experiments.

    Techniques Used: Expressing, Transduction, Construct, FLAG-tag, Flow Cytometry

    The recombinant SARS-CoV-2 GFP/ΔN trVLP can propagate with the help of viral N protein. (A) Experimental scheme. Caco-2 or Caco-2-N cells were infected with SARS-CoV-2 GFP/ΔN for 3h (MOI 0.05), washed, and incubated for an additional 72 h. GFP fluorescence were observed or quantified by microscopy or flow cytometry analysis. Viral RNA was determined by RT-qPCR assay; (B) GFP expression was observed in Caco-2 or Caco-2-N cells using microscopy at indicated time point after inoculation; Representative images from one of three independent experiments. (C) Cell lysates were resolved by SDS-PAGE and probed with anti-Spike and anti-Tubulin antibodies. Representative images from two independent experiments; (D) The total RNAs were extracted and RT-qPCR assays were conducted to determine viral RNA levels. Error bars represent the standard deviations from one of two independent experiments performed in triplicate; (E) RT-PCR analysis of the SARS-CoV-2 GFP/ΔN genome in Caco-2-N cells infected with recombinant virus using a primer set flanking the N region. The expected DNA sized were indicated in each genome, and DNA marker is shown on the left. Representative images from one of two independent experiments; (F-G) Recombinant SARS-CoV-2 GFP/ΔN virus was incubated with indicated doses of neutralizing mAbs against SARS-CoV-2 (1F11 and 2F6) or HIV (VCR01), as well as soluble human ACE2-Fc or F10sFV for 1 h prior to inoculation. The infection was analyzed by GFP expression 2 days later, and the number of positive cells was expressed as a percentage of that for the VRC01 or F10sFV treatment control. Error bars represent the standard deviations from three independent experiments (n=6). ***, P
    Figure Legend Snippet: The recombinant SARS-CoV-2 GFP/ΔN trVLP can propagate with the help of viral N protein. (A) Experimental scheme. Caco-2 or Caco-2-N cells were infected with SARS-CoV-2 GFP/ΔN for 3h (MOI 0.05), washed, and incubated for an additional 72 h. GFP fluorescence were observed or quantified by microscopy or flow cytometry analysis. Viral RNA was determined by RT-qPCR assay; (B) GFP expression was observed in Caco-2 or Caco-2-N cells using microscopy at indicated time point after inoculation; Representative images from one of three independent experiments. (C) Cell lysates were resolved by SDS-PAGE and probed with anti-Spike and anti-Tubulin antibodies. Representative images from two independent experiments; (D) The total RNAs were extracted and RT-qPCR assays were conducted to determine viral RNA levels. Error bars represent the standard deviations from one of two independent experiments performed in triplicate; (E) RT-PCR analysis of the SARS-CoV-2 GFP/ΔN genome in Caco-2-N cells infected with recombinant virus using a primer set flanking the N region. The expected DNA sized were indicated in each genome, and DNA marker is shown on the left. Representative images from one of two independent experiments; (F-G) Recombinant SARS-CoV-2 GFP/ΔN virus was incubated with indicated doses of neutralizing mAbs against SARS-CoV-2 (1F11 and 2F6) or HIV (VCR01), as well as soluble human ACE2-Fc or F10sFV for 1 h prior to inoculation. The infection was analyzed by GFP expression 2 days later, and the number of positive cells was expressed as a percentage of that for the VRC01 or F10sFV treatment control. Error bars represent the standard deviations from three independent experiments (n=6). ***, P

    Techniques Used: Recombinant, Infection, Incubation, Fluorescence, Microscopy, Flow Cytometry, Quantitative RT-PCR, Expressing, SDS Page, Reverse Transcription Polymerase Chain Reaction, Marker

    High throughput screening of antivirals against SARS-CoV-2 infection using trVLP system. (A) Screening of 377 compounds from Topscience Natural Product Library and hits selection. The purple dot line represents the threshold (40%) for positive hit compounds. DMSO (blue) and remdesivir (red) are used as the control for the screening. Each dot represents a single compound, and the green dots represent the promising candidates which exhibited potent antiviral activity without dramatic cytotoxic effect. (B-F) Dose response curves of selected hit compounds. Compounds concentrations are presented in log scale for logarithmic interpolation. Dose response curves were generated using GraphPad Prism software version 7.0. IC 50 values were calculated using Prism software and is representative of one of three independent experiments. Error bars represent the standard deviations from one of three independent experiments performed in triplicate. (G) Comparison of antiviral activity and cytotoxicity of each compound. Selectivity Index (SI), a ratio that compares a drug’s cytotoxicity and antiviral activity was also calculated. n.d.=not detected; n.a.=not applicable.
    Figure Legend Snippet: High throughput screening of antivirals against SARS-CoV-2 infection using trVLP system. (A) Screening of 377 compounds from Topscience Natural Product Library and hits selection. The purple dot line represents the threshold (40%) for positive hit compounds. DMSO (blue) and remdesivir (red) are used as the control for the screening. Each dot represents a single compound, and the green dots represent the promising candidates which exhibited potent antiviral activity without dramatic cytotoxic effect. (B-F) Dose response curves of selected hit compounds. Compounds concentrations are presented in log scale for logarithmic interpolation. Dose response curves were generated using GraphPad Prism software version 7.0. IC 50 values were calculated using Prism software and is representative of one of three independent experiments. Error bars represent the standard deviations from one of three independent experiments performed in triplicate. (G) Comparison of antiviral activity and cytotoxicity of each compound. Selectivity Index (SI), a ratio that compares a drug’s cytotoxicity and antiviral activity was also calculated. n.d.=not detected; n.a.=not applicable.

    Techniques Used: High Throughput Screening Assay, Infection, Selection, Activity Assay, Generated, Software

    Characterization of the genetic stability of SARS-CoV-2 GFP/ΔN trVLP. (A) Detection of the GFP reporter gene during viral passage. RNAs were extracted from the VLP infected cells of P0 to P10 passage, respectively. (B) RT-PCR was performed with a primer pair flanking the N region of ORF8 and 3’UTR. The PCR products were resolved on an agarose gel using electrophoresis. The numbers of time points-samples-passage were denoted on the top of each lane. Representative images from one of three independent experiments; (C-D) RNA-seq coverage of viruse derived reads aligned to SARS-CoV-2 (C) or SARS-CoV-2 GFP/ΔN (D) genome, respectively. (E) Heatmap shows the expression levels of each subgenomic RNA of P1 or P10 trVLP.
    Figure Legend Snippet: Characterization of the genetic stability of SARS-CoV-2 GFP/ΔN trVLP. (A) Detection of the GFP reporter gene during viral passage. RNAs were extracted from the VLP infected cells of P0 to P10 passage, respectively. (B) RT-PCR was performed with a primer pair flanking the N region of ORF8 and 3’UTR. The PCR products were resolved on an agarose gel using electrophoresis. The numbers of time points-samples-passage were denoted on the top of each lane. Representative images from one of three independent experiments; (C-D) RNA-seq coverage of viruse derived reads aligned to SARS-CoV-2 (C) or SARS-CoV-2 GFP/ΔN (D) genome, respectively. (E) Heatmap shows the expression levels of each subgenomic RNA of P1 or P10 trVLP.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, RNA Sequencing Assay, Derivative Assay, Expressing

    Site-specific phosphorylation of N is required to support virus life cycle. (A) Schematics and alignments of N proteins from MERS-CoV, SARS-CoV and SARS-CoV-2. The phosphorylation sites in SARS-CoV-2 N protein were highlighted. (B) Schematic presentation of assessment of N variants function. The trVLP inoculated with Caco-2 cells transduced with N variants, and the cell culture medium were collected to infect the Caco-2-N cells, and GFP expression analyzed by flow cytometry/microscopy or viral subgenomic RNA abundance were determined by RT-qPCR. (C) Western blotting assay was performed to detect the N proteins expression in Caco-2 cells transduced with distinct N genes from SARS-CoV-2, SARS-CoV or MERS-CoV. (D-E) The cell culture medium was collected from SARS-CoV-2 GFP/ΔN trVLP infected Caco-2 cells expressing N from SARS-CoV-2, SARS-CoV or MERS-CoV to infect the naïve Caco-2-N cells. GFP were observed using microscopy and cellular RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. (F) Western blotting assay detected the expression of SARS-CoV-2 N WT or mutants in Caco-2 cells. (G-H) The cell culture medium was collected from SARS-CoV-2 GFP/ΔN trVLP infected Caco-2 cells expressing SARS-CoV-2 N mutants to infect the naïve Caco-2-N cells. GFP were observed using microscopy and cellular RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. (I) GSK-3 inhibitors LiCl or SB216763 treated Caco-2-N cells inoculated with SARS-CoV-2 GFP/ΔN trVLP, the cell culture medium was then inoculated with Caco-2-N cells. RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. Cell viability was evaluated by CellTiter-Glo assay. Error bars (E, H and I) represent the standard deviations from one of three independent experiments performed in triplicate. n.s. no significance; *, P
    Figure Legend Snippet: Site-specific phosphorylation of N is required to support virus life cycle. (A) Schematics and alignments of N proteins from MERS-CoV, SARS-CoV and SARS-CoV-2. The phosphorylation sites in SARS-CoV-2 N protein were highlighted. (B) Schematic presentation of assessment of N variants function. The trVLP inoculated with Caco-2 cells transduced with N variants, and the cell culture medium were collected to infect the Caco-2-N cells, and GFP expression analyzed by flow cytometry/microscopy or viral subgenomic RNA abundance were determined by RT-qPCR. (C) Western blotting assay was performed to detect the N proteins expression in Caco-2 cells transduced with distinct N genes from SARS-CoV-2, SARS-CoV or MERS-CoV. (D-E) The cell culture medium was collected from SARS-CoV-2 GFP/ΔN trVLP infected Caco-2 cells expressing N from SARS-CoV-2, SARS-CoV or MERS-CoV to infect the naïve Caco-2-N cells. GFP were observed using microscopy and cellular RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. (F) Western blotting assay detected the expression of SARS-CoV-2 N WT or mutants in Caco-2 cells. (G-H) The cell culture medium was collected from SARS-CoV-2 GFP/ΔN trVLP infected Caco-2 cells expressing SARS-CoV-2 N mutants to infect the naïve Caco-2-N cells. GFP were observed using microscopy and cellular RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. (I) GSK-3 inhibitors LiCl or SB216763 treated Caco-2-N cells inoculated with SARS-CoV-2 GFP/ΔN trVLP, the cell culture medium was then inoculated with Caco-2-N cells. RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. Cell viability was evaluated by CellTiter-Glo assay. Error bars (E, H and I) represent the standard deviations from one of three independent experiments performed in triplicate. n.s. no significance; *, P

    Techniques Used: Transduction, Cell Culture, Expressing, Flow Cytometry, Microscopy, Quantitative RT-PCR, Western Blot, Infection, Glo Assay

    GFP expression in Caco-2-N cells electroporated with SARS-CoV-2 GFP/ΔN RNA. (A) GFP expression in Caco-2-N cells electroporated with SARS-CoV-2 GFP/ΔN RNA. Caco-2-N cells were electroporated with 20 μg of SARS-CoV-2 GFP/ΔN RNA. From 21h-96h p.t., GFP expression in the cells was observed with microscopy. (B) GFP expression was quantified by flowcytometry at 96h post transfection of the RNA. This experiment was representative of three independent experiments.
    Figure Legend Snippet: GFP expression in Caco-2-N cells electroporated with SARS-CoV-2 GFP/ΔN RNA. (A) GFP expression in Caco-2-N cells electroporated with SARS-CoV-2 GFP/ΔN RNA. Caco-2-N cells were electroporated with 20 μg of SARS-CoV-2 GFP/ΔN RNA. From 21h-96h p.t., GFP expression in the cells was observed with microscopy. (B) GFP expression was quantified by flowcytometry at 96h post transfection of the RNA. This experiment was representative of three independent experiments.

    Techniques Used: Expressing, Microscopy, Transfection

    Reconstitution of functional N protein by intein-mediated protein splicing. (A) Scheme depicting of intein-mediated protein trans-splicing to reconstitute full length N protein. (B) Western blot (WB) analysis of lysates from Caco-2 cells transduced with either full-length N or intein-N lentiviruses. The star indicates the full-length N protein. The WB is representative of three independent experiments. (C) GFP fluorescence in Caco-2-N cells infected cell culture medium (containing SARS-CoV-2 GFP/ΔN progeny) collected from each Caco-2-N int cells which was inoculated with SARS-CoV-2 GFP/ΔN trVLP at 2 days of culture. The image is representative of n=4. (D) Cells were harvested to quantify GFP expression by flow cytometry analysis, and (E) Subgenomic RNA of E were determined by RT-qPCR assay. Error bars represent the standard deviations from one of three independent experiments performed in triplicate. *, P
    Figure Legend Snippet: Reconstitution of functional N protein by intein-mediated protein splicing. (A) Scheme depicting of intein-mediated protein trans-splicing to reconstitute full length N protein. (B) Western blot (WB) analysis of lysates from Caco-2 cells transduced with either full-length N or intein-N lentiviruses. The star indicates the full-length N protein. The WB is representative of three independent experiments. (C) GFP fluorescence in Caco-2-N cells infected cell culture medium (containing SARS-CoV-2 GFP/ΔN progeny) collected from each Caco-2-N int cells which was inoculated with SARS-CoV-2 GFP/ΔN trVLP at 2 days of culture. The image is representative of n=4. (D) Cells were harvested to quantify GFP expression by flow cytometry analysis, and (E) Subgenomic RNA of E were determined by RT-qPCR assay. Error bars represent the standard deviations from one of three independent experiments performed in triplicate. *, P

    Techniques Used: Functional Assay, Western Blot, Transduction, Fluorescence, Infection, Cell Culture, Expressing, Flow Cytometry, Quantitative RT-PCR

    Production of SARS-CoV-2 GFP/ΔN trVLP. (A) The top rows show genetic organizations of the SARS-CoV-2 and SARS-CoV-2 GFP/ΔN genomes. The ORF of N is replaced with reporter gene (GFP here). The cDNA of SARS-CoV-2 GFP/ΔN genome was divided into five fragments designated as Fragment A, B, C, D and E, which could be obtained by PCR (B). Each cDNA fragment was flanked by a class IIS restriction endonuclease site (BsaI or BsmBI) and the nucleotide sequences and locations of the cohesive overhangs are indicated. The fragment cDNA were digested and purified for directed assembly of SARS-CoV-2 GFP/ΔN cDNA (see C panel, and the star indicates the genome-length cDNA), which served as the template for in vitro transcription to generate viral RNA genome (see D panel, and the star indicates the genome-length RNA transcript). The viral genomic RNAs were electroporated into Caco-2-N cells. After 3 days, the supernatant was collected and inoculated with Caco-2 or Caco-2-N cells. (E). Western blotting assay was performed to detect the expression of N proteins and ACE2 in Caco-2-N cells, Vero E6 and Vero E6-N cells.
    Figure Legend Snippet: Production of SARS-CoV-2 GFP/ΔN trVLP. (A) The top rows show genetic organizations of the SARS-CoV-2 and SARS-CoV-2 GFP/ΔN genomes. The ORF of N is replaced with reporter gene (GFP here). The cDNA of SARS-CoV-2 GFP/ΔN genome was divided into five fragments designated as Fragment A, B, C, D and E, which could be obtained by PCR (B). Each cDNA fragment was flanked by a class IIS restriction endonuclease site (BsaI or BsmBI) and the nucleotide sequences and locations of the cohesive overhangs are indicated. The fragment cDNA were digested and purified for directed assembly of SARS-CoV-2 GFP/ΔN cDNA (see C panel, and the star indicates the genome-length cDNA), which served as the template for in vitro transcription to generate viral RNA genome (see D panel, and the star indicates the genome-length RNA transcript). The viral genomic RNAs were electroporated into Caco-2-N cells. After 3 days, the supernatant was collected and inoculated with Caco-2 or Caco-2-N cells. (E). Western blotting assay was performed to detect the expression of N proteins and ACE2 in Caco-2-N cells, Vero E6 and Vero E6-N cells.

    Techniques Used: Polymerase Chain Reaction, Purification, In Vitro, Western Blot, Expressing

    Characterization of the genetic stability of SARS-CoV-2 GFP/ΔN virus. (A) RT-PCR products from P10 virus infected cell passage were cloned into pEASY-Blunt vector, and 12 colonies were randomly chosen for DNA sequences analysis. Multiple deletions were detected in the amplicon. (B) Categories of mapped reads from P1 and P10 virus infected Caco-2-N cells. (C) Canonical discontinuous transcription (top) that is mediated by TRS-L (TRS in the leader) and TRS-B (TRS in the body). Quantification of junction-reads from canonical discontinuous transcripts post P1 and P10 virus infection.
    Figure Legend Snippet: Characterization of the genetic stability of SARS-CoV-2 GFP/ΔN virus. (A) RT-PCR products from P10 virus infected cell passage were cloned into pEASY-Blunt vector, and 12 colonies were randomly chosen for DNA sequences analysis. Multiple deletions were detected in the amplicon. (B) Categories of mapped reads from P1 and P10 virus infected Caco-2-N cells. (C) Canonical discontinuous transcription (top) that is mediated by TRS-L (TRS in the leader) and TRS-B (TRS in the body). Quantification of junction-reads from canonical discontinuous transcripts post P1 and P10 virus infection.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Infection, Clone Assay, Plasmid Preparation, Amplification

    Inhibition of recombinant SARS-CoV-2 GFP/ΔN trVLP infection by IFN and antivirals. (A) IFN-β pretreated Caco-2-N int cells were subsequently infected with trVLP and cells were subjected to flow cytometry analysis for quantify the GFP fluorescence at 2 days post-infection. Error bars represent the standard deviations from three independent experiments (n=6). (B-E) Antiviral effect of remdesivir, GC376, lopinavir and ritonavir. The drug treated cells were infected with trVLP and GFP fluorescence was quantified at 48h after infection. The cytotoxic effect of each drug at indicated concentrations were determined by CellTiter-Glo cell viability assay. The virus infection or cytotoxicity is plotted versus compound concentration (n=3 biological replicates for all compounds). The black dots indicate replicate measurements, and the black lines indicate dose-response curve fits. The red dots indicate cytotoxicity. IC 50 values were calculated using Prism software and is representative of one of three independent experiments performed in triplicate. Three independent experiments had similar results. (F) Comparison of antiviral activity and cytotoxicity of each compound. Selectivity Index (SI), a ratio that compares a drug’s cytotoxicity and antiviral activity was also calculated. n.d.=not detected; n.a.=not applicable.
    Figure Legend Snippet: Inhibition of recombinant SARS-CoV-2 GFP/ΔN trVLP infection by IFN and antivirals. (A) IFN-β pretreated Caco-2-N int cells were subsequently infected with trVLP and cells were subjected to flow cytometry analysis for quantify the GFP fluorescence at 2 days post-infection. Error bars represent the standard deviations from three independent experiments (n=6). (B-E) Antiviral effect of remdesivir, GC376, lopinavir and ritonavir. The drug treated cells were infected with trVLP and GFP fluorescence was quantified at 48h after infection. The cytotoxic effect of each drug at indicated concentrations were determined by CellTiter-Glo cell viability assay. The virus infection or cytotoxicity is plotted versus compound concentration (n=3 biological replicates for all compounds). The black dots indicate replicate measurements, and the black lines indicate dose-response curve fits. The red dots indicate cytotoxicity. IC 50 values were calculated using Prism software and is representative of one of three independent experiments performed in triplicate. Three independent experiments had similar results. (F) Comparison of antiviral activity and cytotoxicity of each compound. Selectivity Index (SI), a ratio that compares a drug’s cytotoxicity and antiviral activity was also calculated. n.d.=not detected; n.a.=not applicable.

    Techniques Used: Inhibition, Recombinant, Infection, Flow Cytometry, Fluorescence, Viability Assay, Concentration Assay, Software, Activity Assay

    Identification of host factors associated with N protein and phosphorylation on N protein by mass spectrometry. (A) Flag tagged N protein was immunoprecipitated from Caco-2-N cells infected with recombinant SARS-CoV-2 GFP/ΔN trVLP using Flag antibody, and the proteins were analyzed on SDS-PAGE gel. The proteins were visualized by Coomassie blue staining. N, G3BP1 and G3BP2 were labelled. (B) Phosphorylated peptides of N protein derived from Caco-2-N cells. Caco-2-N cells in which N was C-terminal Flag-tagged were collected and cell lysates were immunoprecipitated with anti-Flag coupled beads. Phosphorylated peptides of the immunoprecipitates were analyzed by mass spectrometry.
    Figure Legend Snippet: Identification of host factors associated with N protein and phosphorylation on N protein by mass spectrometry. (A) Flag tagged N protein was immunoprecipitated from Caco-2-N cells infected with recombinant SARS-CoV-2 GFP/ΔN trVLP using Flag antibody, and the proteins were analyzed on SDS-PAGE gel. The proteins were visualized by Coomassie blue staining. N, G3BP1 and G3BP2 were labelled. (B) Phosphorylated peptides of N protein derived from Caco-2-N cells. Caco-2-N cells in which N was C-terminal Flag-tagged were collected and cell lysates were immunoprecipitated with anti-Flag coupled beads. Phosphorylated peptides of the immunoprecipitates were analyzed by mass spectrometry.

    Techniques Used: Mass Spectrometry, Immunoprecipitation, Infection, Recombinant, SDS Page, Staining, Derivative Assay

    6) Product Images from "Binding strength and hydrogen bond numbers between Covid-19 RBD and HVR of antibody"

    Article Title: Binding strength and hydrogen bond numbers between Covid-19 RBD and HVR of antibody

    Journal: bioRxiv

    doi: 10.1101/2020.12.21.423787

    (a) The FTIR scan of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2), obtained from more concentrated samples, showing a visible 750 cm -1 bands at 27-29°C (red circle), exclusively associated with the proteins, but not found in the PBS solvent, attributed to the possible Van der Waals bonding between the large pendant group such as phenyl. (b) Similar FTIR result of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1) obtained from more concentrated samples, showing a visible 750 cm -1 bands at 27-29°C (red circle). (c) Similar FTIR result of SARS-CoV-2 spike protein alone, obtained from more concentrated samples, showing a visible 750 cm -1 bands at 27-29°C (red circle). (d) The infrared absorption of 750cm -1 under variable temperature of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2), SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1), and the spike protein alone, obtained from the difference between the peak near 750cm -1 and the trough near 725cm -1 , after subtracting that of the buffer solution. The associated error bars were estimated by combining the instrumental error, and the possible deviation involved in the computation.
    Figure Legend Snippet: (a) The FTIR scan of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2), obtained from more concentrated samples, showing a visible 750 cm -1 bands at 27-29°C (red circle), exclusively associated with the proteins, but not found in the PBS solvent, attributed to the possible Van der Waals bonding between the large pendant group such as phenyl. (b) Similar FTIR result of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1) obtained from more concentrated samples, showing a visible 750 cm -1 bands at 27-29°C (red circle). (c) Similar FTIR result of SARS-CoV-2 spike protein alone, obtained from more concentrated samples, showing a visible 750 cm -1 bands at 27-29°C (red circle). (d) The infrared absorption of 750cm -1 under variable temperature of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2), SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1), and the spike protein alone, obtained from the difference between the peak near 750cm -1 and the trough near 725cm -1 , after subtracting that of the buffer solution. The associated error bars were estimated by combining the instrumental error, and the possible deviation involved in the computation.

    Techniques Used:

    (a) The FTIR scan of SARS-CoV-2 spike protein (S protein) in PBS solution, for the wavenumber range of 1400-1800cm -1 . (b) The FTIR scan of SARS-CoV-2 antibody (antibody 2) in PBS solution, for the wavenumber range of 1400-1800cm -1 . (c) The FTIR scan of SARS-CoV-1 antibody (antibody 1) in PBS solution, for the wavenumber range of 1400-1800cm -1 . (d) The infrared absorption of 1550cm -1 under variable temperature, of SARS-CoV-2 spike protein, SARS-CoV-2 antibody, and SARS-CoV-1 antibody, respectively, given by the difference between the crest near 1550cm -1 , and the trough near 1480cm -1 . The associated error bars were estimated by combining the instrumental error, and the possible deviation involved in the computation. All 3 proteins followed a similar trend in temperature, which is attributed to the thermal agitation, when the likelihood increases for such binding sites to pair, either between the neighbouring proteins, or amongst various strands within a protein, or even along a protein strand. (e) Schematic illustration of the hydrogen bonding between carboxyl and amino groups within a protein strand, showing the intra-molecular binding of the quaternary structure of proteins, when the Van der Waals bonds between large pendant groups, such as phenyl, were disrupted, which causes unfolding and exposing more bonding sites.
    Figure Legend Snippet: (a) The FTIR scan of SARS-CoV-2 spike protein (S protein) in PBS solution, for the wavenumber range of 1400-1800cm -1 . (b) The FTIR scan of SARS-CoV-2 antibody (antibody 2) in PBS solution, for the wavenumber range of 1400-1800cm -1 . (c) The FTIR scan of SARS-CoV-1 antibody (antibody 1) in PBS solution, for the wavenumber range of 1400-1800cm -1 . (d) The infrared absorption of 1550cm -1 under variable temperature, of SARS-CoV-2 spike protein, SARS-CoV-2 antibody, and SARS-CoV-1 antibody, respectively, given by the difference between the crest near 1550cm -1 , and the trough near 1480cm -1 . The associated error bars were estimated by combining the instrumental error, and the possible deviation involved in the computation. All 3 proteins followed a similar trend in temperature, which is attributed to the thermal agitation, when the likelihood increases for such binding sites to pair, either between the neighbouring proteins, or amongst various strands within a protein, or even along a protein strand. (e) Schematic illustration of the hydrogen bonding between carboxyl and amino groups within a protein strand, showing the intra-molecular binding of the quaternary structure of proteins, when the Van der Waals bonds between large pendant groups, such as phenyl, were disrupted, which causes unfolding and exposing more bonding sites.

    Techniques Used: Binding Assay

    (a) Schematic illustration of the hydrogen bonding between carboxyl and amino groups of the protein strands, representing a typical intermolecular binding for the secondary structure of proteins. (b) The FTIR apparatus, the ATR model has been adopted to improve the signal noise ratio, which offers 0.1% error margin in the absorption measurement, and 0.5cm -1 along the wavenumber scan. To facilitate the possible temperature variation, an electric heating element, and a thermometer were attached to the metallic plate of the ATR assembly, which also acts as an ideal heat sink. Samples of antibodies and spike proteins were mixed together, and undergone FTIR spectroscopic examination conditioned by variable temperatures. (c) The FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2) in PBS solution. Comparing the FTIR signal of the PBS solvent, it indicates that 1550 cm -1 was the only absorption band exclusively associated with the proteins, but not overshadowed by the PBS solvent. (d) The same FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1) in PBS solution.
    Figure Legend Snippet: (a) Schematic illustration of the hydrogen bonding between carboxyl and amino groups of the protein strands, representing a typical intermolecular binding for the secondary structure of proteins. (b) The FTIR apparatus, the ATR model has been adopted to improve the signal noise ratio, which offers 0.1% error margin in the absorption measurement, and 0.5cm -1 along the wavenumber scan. To facilitate the possible temperature variation, an electric heating element, and a thermometer were attached to the metallic plate of the ATR assembly, which also acts as an ideal heat sink. Samples of antibodies and spike proteins were mixed together, and undergone FTIR spectroscopic examination conditioned by variable temperatures. (c) The FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2) in PBS solution. Comparing the FTIR signal of the PBS solvent, it indicates that 1550 cm -1 was the only absorption band exclusively associated with the proteins, but not overshadowed by the PBS solvent. (d) The same FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1) in PBS solution.

    Techniques Used: Binding Assay

    (a) Detailed FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2) in PBS solution, within the wavenumber range of 1400-1800cm -1 . (b) Detailed FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1) in PBS solution, in the wavenumber range of 1400-1800cm -1 . (c) The infrared absorptions of 1550cm -1 under variable temperature of the sample combination of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody, versus the sample combination of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody, obtained from the difference between the peak near 1550cm -1 and the trough near 1480cm -1 , after subtracting that of the buffer solution. The associated error bars were estimated by combining the instrumental error, and the possible deviation involved in the computation. A strong temperature dependence is observed, where the bonding number of the antibody is enhanced sharply beyond 31°C, rather than at the usual room temperature. (d) The possible temperature influence on the structure of spike protein and antibodies, where only the top part of the IgM, similar to IgG, was depicted: a higher temperature increases the probability of protein quaternary structure unfolding, and exposes more binding sites, hence more bonds.
    Figure Legend Snippet: (a) Detailed FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2) in PBS solution, within the wavenumber range of 1400-1800cm -1 . (b) Detailed FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1) in PBS solution, in the wavenumber range of 1400-1800cm -1 . (c) The infrared absorptions of 1550cm -1 under variable temperature of the sample combination of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody, versus the sample combination of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody, obtained from the difference between the peak near 1550cm -1 and the trough near 1480cm -1 , after subtracting that of the buffer solution. The associated error bars were estimated by combining the instrumental error, and the possible deviation involved in the computation. A strong temperature dependence is observed, where the bonding number of the antibody is enhanced sharply beyond 31°C, rather than at the usual room temperature. (d) The possible temperature influence on the structure of spike protein and antibodies, where only the top part of the IgM, similar to IgG, was depicted: a higher temperature increases the probability of protein quaternary structure unfolding, and exposes more binding sites, hence more bonds.

    Techniques Used: Binding Assay

    7) Product Images from "Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry"

    Article Title: Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-78895-x

    BLI-ISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a , b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:8 dilution. Bars and dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( a ) The Total Antibody Binding signal is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. ( b ) The Detection signal is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( c ) Dilution series BLI-ISA from representative strong (SP7) and moderate (SP8) seropositive samples. ( d ) Dilution series BLI-ISA from the weakest seropositive sample (SP3) compared to seronegative plasma samples.
    Figure Legend Snippet: BLI-ISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a , b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:8 dilution. Bars and dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( a ) The Total Antibody Binding signal is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. ( b ) The Detection signal is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( c ) Dilution series BLI-ISA from representative strong (SP7) and moderate (SP8) seropositive samples. ( d ) Dilution series BLI-ISA from the weakest seropositive sample (SP3) compared to seronegative plasma samples.

    Techniques Used: Standard Deviation, Binding Assay

    ELISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a ) Single-dilution ELISA to evaluate the presence of RBD-reactive human IgG in pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:50 dilution. Samples were evaluated in biological duplicates and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( b ) Dilution series ELISA was performed to quantitate RBD-reactive human IgG in plasma. Samples were evaluated in biological duplicates. Dashed curves represent fit lines from a four-parameter logistic regression applied over each series. ( c ) Data from ( b ) plotted as area-under-the-curve (AUC).
    Figure Legend Snippet: ELISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a ) Single-dilution ELISA to evaluate the presence of RBD-reactive human IgG in pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:50 dilution. Samples were evaluated in biological duplicates and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( b ) Dilution series ELISA was performed to quantitate RBD-reactive human IgG in plasma. Samples were evaluated in biological duplicates. Dashed curves represent fit lines from a four-parameter logistic regression applied over each series. ( c ) Data from ( b ) plotted as area-under-the-curve (AUC).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Standard Deviation

    BLI-ISA evaluation of plasma antibodies to SARS-CoV-2 prefusion Spike and plasma IgA to SARS-CoV-2 spike RBD. ( a ) Single-dilution BLI-ISA to evaluate the presence of prefusion Spike-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples. The Total Antibody Binding signal (left) is measured when prefusion Spike-His-loaded HIS1K biosensors are dipped into plasma samples. The Detection signal (right) is measured when prefusion Spike-His-loaded HIS1K biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the samples. The Total Antibody Binding signal (left) is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. The Detection signal (right) is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgA. The SP7 dot is colored pink to indicate that this sample had a negative signal (value in parentheses) in the Detection step. All assays were performed with plasma at a 1:8 dilution. Dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean.
    Figure Legend Snippet: BLI-ISA evaluation of plasma antibodies to SARS-CoV-2 prefusion Spike and plasma IgA to SARS-CoV-2 spike RBD. ( a ) Single-dilution BLI-ISA to evaluate the presence of prefusion Spike-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples. The Total Antibody Binding signal (left) is measured when prefusion Spike-His-loaded HIS1K biosensors are dipped into plasma samples. The Detection signal (right) is measured when prefusion Spike-His-loaded HIS1K biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the samples. The Total Antibody Binding signal (left) is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. The Detection signal (right) is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgA. The SP7 dot is colored pink to indicate that this sample had a negative signal (value in parentheses) in the Detection step. All assays were performed with plasma at a 1:8 dilution. Dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean.

    Techniques Used: Binding Assay, Standard Deviation

    8) Product Images from "Distinct conformational states of SARS-CoV-2 spike protein"

    Article Title: Distinct conformational states of SARS-CoV-2 spike protein

    Journal: bioRxiv

    doi: 10.1101/2020.05.16.099317

    A model for structural rearrangements of SARS-Cov-2 S protein. (A) Structural changes independent of a target cell. We suggest that both the prefusion and postfusion spikes are present on the surface of mature virion and the ratio between them may vary (diagram of virion). The postfusion spikes on the virion are formed by S2 after S1 dissociates in the absence of ACE2. (B) ACE2-dependent structural rearrangements. Structural transition from the prefusion to postfusion conformation inducing membrane fusion likely proceeds stepwise as follows: 1) FPPR clamps down RBD through CTD1 in the prefusion S trimer, but it occasionally flips out of position and allows an RBD to sample the up conformation. 2) RBD binding to ACE2 creates a flexible FPPR that enables exposure of the S2’ cleavage site immediately upstream of the adjacent fusion peptide (FP). Cleavage at the S2’ site, and perhaps also the S1/S2 site, releases the structural constraints on the fusion peptide and initiates a cascade of refolding events in S2, probably accompanied by complete dissociation of S1. 3) Formation of the long central three-stranded coiled-coil and folding back of HR2. 4) Formation of the postfusion structure of S2 that brings the two membranes together, facilitating formation of a fusion pore and viral entry.
    Figure Legend Snippet: A model for structural rearrangements of SARS-Cov-2 S protein. (A) Structural changes independent of a target cell. We suggest that both the prefusion and postfusion spikes are present on the surface of mature virion and the ratio between them may vary (diagram of virion). The postfusion spikes on the virion are formed by S2 after S1 dissociates in the absence of ACE2. (B) ACE2-dependent structural rearrangements. Structural transition from the prefusion to postfusion conformation inducing membrane fusion likely proceeds stepwise as follows: 1) FPPR clamps down RBD through CTD1 in the prefusion S trimer, but it occasionally flips out of position and allows an RBD to sample the up conformation. 2) RBD binding to ACE2 creates a flexible FPPR that enables exposure of the S2’ cleavage site immediately upstream of the adjacent fusion peptide (FP). Cleavage at the S2’ site, and perhaps also the S1/S2 site, releases the structural constraints on the fusion peptide and initiates a cascade of refolding events in S2, probably accompanied by complete dissociation of S1. 3) Formation of the long central three-stranded coiled-coil and folding back of HR2. 4) Formation of the postfusion structure of S2 that brings the two membranes together, facilitating formation of a fusion pore and viral entry.

    Techniques Used: Binding Assay

    Cryo-EM structure of the SARS-CoV-2 S2 in the postfusion conformation. (A) The structure of the S2 trimer was modeled based on a 3.3Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of the S2 trimer in the postfusion conformation shown in ribbon diagram. Various structural components in the color scheme shown in Fig. 1A include HR1, heptad repeat 1; CH, central helix region; CD, connector domain; and HR1, heptad repeat 2. The S2’ cleavage site is in a disordered loop between Ile770 and Thr912. A possible location of the S2 N-terminus (S1/S2 cleavage site) is also indicated. (C) A low resolution map showing the density pattern for 5 N-linked glycans, with almost equal spacing along the long axis.
    Figure Legend Snippet: Cryo-EM structure of the SARS-CoV-2 S2 in the postfusion conformation. (A) The structure of the S2 trimer was modeled based on a 3.3Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of the S2 trimer in the postfusion conformation shown in ribbon diagram. Various structural components in the color scheme shown in Fig. 1A include HR1, heptad repeat 1; CH, central helix region; CD, connector domain; and HR1, heptad repeat 2. The S2’ cleavage site is in a disordered loop between Ile770 and Thr912. A possible location of the S2 N-terminus (S1/S2 cleavage site) is also indicated. (C) A low resolution map showing the density pattern for 5 N-linked glycans, with almost equal spacing along the long axis.

    Techniques Used:

    Selected new features of the SARS-CoV-2 prefusion S trimer. (A) N-terminal segment of S protein. The N-terminus is at residue Gln14 after cleavage of the signal peptide. Cys15 forms a disulfide bond with Cys136. We observed good density for the N-linked glycan at Asn17. (B) A segment immediately downstream of the fusion peptide, while disordered in the stabilized soluble S ectodomain trimer structure, forms a tightly packed structure, designated FPPR for the fusion peptide proximal region, abutting CTD1. The newly identified FPPR structure would clash with CTD1 in the RBD up conformation. Various domains are shown in the color scheme in Fig. 2B . The structure of the soluble S trimer with one RBD in the up conformation (PDB ID: 6vyb) is shown in gray. In the box, a close-up view of the FPPR with adjacent fusion peptide in both surface representation and stick model. (C) The SARS-CoV-2 prefusion S trimer, viewed along the threefold axis, is superposed on the structure of the stabilized soluble S ectodomain trimer in the closed conformation with all three RBDs in the down conformation (PDB ID: 6vxx). While the S2 region is well aligned, there is a significant shift (e.g. ∼10Å between two Ala123 residues) in S1. (D) Impact of the proline mutations introduced at residues 986 and 987 to stabilize the prefusion conformation. K986P mutation removes a salt bridge between Lys986 of one protomer and either Asp426 or Asp427 of another protomer in the trimer interface.
    Figure Legend Snippet: Selected new features of the SARS-CoV-2 prefusion S trimer. (A) N-terminal segment of S protein. The N-terminus is at residue Gln14 after cleavage of the signal peptide. Cys15 forms a disulfide bond with Cys136. We observed good density for the N-linked glycan at Asn17. (B) A segment immediately downstream of the fusion peptide, while disordered in the stabilized soluble S ectodomain trimer structure, forms a tightly packed structure, designated FPPR for the fusion peptide proximal region, abutting CTD1. The newly identified FPPR structure would clash with CTD1 in the RBD up conformation. Various domains are shown in the color scheme in Fig. 2B . The structure of the soluble S trimer with one RBD in the up conformation (PDB ID: 6vyb) is shown in gray. In the box, a close-up view of the FPPR with adjacent fusion peptide in both surface representation and stick model. (C) The SARS-CoV-2 prefusion S trimer, viewed along the threefold axis, is superposed on the structure of the stabilized soluble S ectodomain trimer in the closed conformation with all three RBDs in the down conformation (PDB ID: 6vxx). While the S2 region is well aligned, there is a significant shift (e.g. ∼10Å between two Ala123 residues) in S1. (D) Impact of the proline mutations introduced at residues 986 and 987 to stabilize the prefusion conformation. K986P mutation removes a salt bridge between Lys986 of one protomer and either Asp426 or Asp427 of another protomer in the trimer interface.

    Techniques Used: Mutagenesis

    Preparation of a full-length SARS-CoV-2 spike protein. (A) Schematic representation of the expression construct of full-length SARS-CoV-2 spike (S) protein. Segments of S1 and S2 include: NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; S1/S2, S1/S2 cleavage site; S2’, S2’ cleavage site; FP, fusion peptide; FFPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane anchor; CT, cytoplasmic tail; and tree-like symbols for glycans. A strep-tag was fused to the C-terminus of S protein by a flexible linker. (B) The purified S protein was resolved by gel-filtration chromatography on a Superose 6 column in the presence of detergent NP-40. The molecular weight standards include thyoglobulin (670 kDa), γ-globulin (158 kDa) and ovalbumin (44 kDa). Three major peaks (peak I-III) contain the S protein. (C) Load sample and peak fractions from (B) were analyzed by Coomassie stained SDS-PAGE. Labeled bands were confirmed by western blot (S, S1 and S2) or protein sequencing (S2 and Cont; S and S1 bands did not gave any meaningful results probably due to a blocked N-terminus). Cont, copurified contaminating protein, identified as endoplasmic reticulum chaperone BiP precursor by N-terminal sequencing. Representative images and 2D averages by negative stain EM of three peak fractions are also shown. The box size of 2D averages is ∼510Å.
    Figure Legend Snippet: Preparation of a full-length SARS-CoV-2 spike protein. (A) Schematic representation of the expression construct of full-length SARS-CoV-2 spike (S) protein. Segments of S1 and S2 include: NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; S1/S2, S1/S2 cleavage site; S2’, S2’ cleavage site; FP, fusion peptide; FFPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane anchor; CT, cytoplasmic tail; and tree-like symbols for glycans. A strep-tag was fused to the C-terminus of S protein by a flexible linker. (B) The purified S protein was resolved by gel-filtration chromatography on a Superose 6 column in the presence of detergent NP-40. The molecular weight standards include thyoglobulin (670 kDa), γ-globulin (158 kDa) and ovalbumin (44 kDa). Three major peaks (peak I-III) contain the S protein. (C) Load sample and peak fractions from (B) were analyzed by Coomassie stained SDS-PAGE. Labeled bands were confirmed by western blot (S, S1 and S2) or protein sequencing (S2 and Cont; S and S1 bands did not gave any meaningful results probably due to a blocked N-terminus). Cont, copurified contaminating protein, identified as endoplasmic reticulum chaperone BiP precursor by N-terminal sequencing. Representative images and 2D averages by negative stain EM of three peak fractions are also shown. The box size of 2D averages is ∼510Å.

    Techniques Used: Expressing, Construct, Binding Assay, Strep-tag, Purification, Filtration, Chromatography, Molecular Weight, Staining, SDS Page, Labeling, Western Blot, Sequencing

    Cryo-EM structure of the SARS-CoV-2 S protein in the prefusion conformation. (A) The structure of the S trimer was modeled based on a 3.1Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of S protein in the prefusion conformation shown in ribbon representation. Various structural components in the color scheme shown in Fig. 1A include NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; and CD, connector domain. N-terminus, S1/S2 cleavage site and S2’ cleavage site are indicated.
    Figure Legend Snippet: Cryo-EM structure of the SARS-CoV-2 S protein in the prefusion conformation. (A) The structure of the S trimer was modeled based on a 3.1Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of S protein in the prefusion conformation shown in ribbon representation. Various structural components in the color scheme shown in Fig. 1A include NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; and CD, connector domain. N-terminus, S1/S2 cleavage site and S2’ cleavage site are indicated.

    Techniques Used: Binding Assay

    9) Product Images from "Presence of antibodies against SARS-CoV-2 spike protein in bovine whey IgG enriched fraction"

    Article Title: Presence of antibodies against SARS-CoV-2 spike protein in bovine whey IgG enriched fraction

    Journal: International Dairy Journal

    doi: 10.1016/j.idairyj.2021.105002

    Bovine IgG enriched fraction containing IgG against SARS-CoV-2 assessed by direct enzyme-linked immunosorbent assays (ELISA) using a partial-length of recombinant SARS-CoV-2 S (aa 177–512, 288–512, 348–578, 387–516 and 408–664), full-recombinant SARS-CoV-2 N (aa 1-419) and partial-length of recombinant SARS-CoV-2 N (aa 1–120, 111–220, 1–220 and 210–419) ( Fig.1 ) as coating antigens. Two different lots of bovine IgG enriched fraction prepared in 2019 and 2018 were used (2a and 2b, respectively): Image 1 , 0.003 μg mL -1 ; Image 2 , 0.03 μg mL -1 ; Image 3 , 0.3 μg mL -1 ; Image 4 , 3 μg mL -1 ; Image 5 , 30 μg mL -1 . A picture of a representative ELISA result is shown in 2c.
    Figure Legend Snippet: Bovine IgG enriched fraction containing IgG against SARS-CoV-2 assessed by direct enzyme-linked immunosorbent assays (ELISA) using a partial-length of recombinant SARS-CoV-2 S (aa 177–512, 288–512, 348–578, 387–516 and 408–664), full-recombinant SARS-CoV-2 N (aa 1-419) and partial-length of recombinant SARS-CoV-2 N (aa 1–120, 111–220, 1–220 and 210–419) ( Fig.1 ) as coating antigens. Two different lots of bovine IgG enriched fraction prepared in 2019 and 2018 were used (2a and 2b, respectively): Image 1 , 0.003 μg mL -1 ; Image 2 , 0.03 μg mL -1 ; Image 3 , 0.3 μg mL -1 ; Image 4 , 3 μg mL -1 ; Image 5 , 30 μg mL -1 . A picture of a representative ELISA result is shown in 2c.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Recombinant

    Competitive inhibition ELISA of the bovine IgG enriched fraction (IgG 0.3 μg mL -1 ), incubated with one of three peptides of S protein of SARS-CoV-2 ( Image 7 , aa 382–401; Image 8 , aa 427–446; Image 9 , aa 502–520) at concentrations 0.001, 0.01, 0,1, 1, and 10 μg mL -1 , the remaining free IgG against the S protein of SARS-CoV-2 was assayed by direct ELISA, using plates coated with the peptide corresponding to aa 288–512 of S protein of SARS-CoV-2. Two lots of bovine IgG enriched fraction prepared in 2019 ( Image 10 ) and 2018 ( Image 11 ) were tested.
    Figure Legend Snippet: Competitive inhibition ELISA of the bovine IgG enriched fraction (IgG 0.3 μg mL -1 ), incubated with one of three peptides of S protein of SARS-CoV-2 ( Image 7 , aa 382–401; Image 8 , aa 427–446; Image 9 , aa 502–520) at concentrations 0.001, 0.01, 0,1, 1, and 10 μg mL -1 , the remaining free IgG against the S protein of SARS-CoV-2 was assayed by direct ELISA, using plates coated with the peptide corresponding to aa 288–512 of S protein of SARS-CoV-2. Two lots of bovine IgG enriched fraction prepared in 2019 ( Image 10 ) and 2018 ( Image 11 ) were tested.

    Techniques Used: Inhibition, Enzyme-linked Immunosorbent Assay, Incubation, Direct ELISA

    Determination of epitopes by direct ELISA using nine peptides of SARS-CoV-2 S protein, corresponding to aa 382–401, 397–416, 427–446, 442–461, 457–476, 472–491, 487–506 and 502–520, with plates coated with a recombinant protein covering the RBD of SARS-CoV-2 S protein. Two lots of bovine IgG enriched fraction prepared in 2019 and 2018 (3a and 3b, respectively), were tested: Image 6 , 0.3 μg mL -1 ; Image 2 , 3 μg mL -1 ; Image 3 , 30 μg mL -1 .
    Figure Legend Snippet: Determination of epitopes by direct ELISA using nine peptides of SARS-CoV-2 S protein, corresponding to aa 382–401, 397–416, 427–446, 442–461, 457–476, 472–491, 487–506 and 502–520, with plates coated with a recombinant protein covering the RBD of SARS-CoV-2 S protein. Two lots of bovine IgG enriched fraction prepared in 2019 and 2018 (3a and 3b, respectively), were tested: Image 6 , 0.3 μg mL -1 ; Image 2 , 3 μg mL -1 ; Image 3 , 30 μg mL -1 .

    Techniques Used: Direct ELISA, Recombinant

    Overall topology of (a) SARS-CoV-2 spike protein (S) and five regions of recombinant SARS-CoV-2 S and (b) SARS-CoV-2 nucleocapsid protein (N) and regions of recombinant SARS-CoV-2 N. NTD, N-terminal domain; CTD, C-terminal domain; RBD, receptor binding domain; RDM, receptor binding motif; SD1, subdomain 1; SD2, subdomain 2; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane region; IC, intracellular domain.
    Figure Legend Snippet: Overall topology of (a) SARS-CoV-2 spike protein (S) and five regions of recombinant SARS-CoV-2 S and (b) SARS-CoV-2 nucleocapsid protein (N) and regions of recombinant SARS-CoV-2 N. NTD, N-terminal domain; CTD, C-terminal domain; RBD, receptor binding domain; RDM, receptor binding motif; SD1, subdomain 1; SD2, subdomain 2; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane region; IC, intracellular domain.

    Techniques Used: Recombinant, Binding Assay

    10) Product Images from "Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry"

    Article Title: Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-78895-x

    BLI-ISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a , b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:8 dilution. Bars and dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( a ) The Total Antibody Binding signal is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. ( b ) The Detection signal is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( c ) Dilution series BLI-ISA from representative strong (SP7) and moderate (SP8) seropositive samples. ( d ) Dilution series BLI-ISA from the weakest seropositive sample (SP3) compared to seronegative plasma samples.
    Figure Legend Snippet: BLI-ISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a , b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:8 dilution. Bars and dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( a ) The Total Antibody Binding signal is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. ( b ) The Detection signal is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( c ) Dilution series BLI-ISA from representative strong (SP7) and moderate (SP8) seropositive samples. ( d ) Dilution series BLI-ISA from the weakest seropositive sample (SP3) compared to seronegative plasma samples.

    Techniques Used: Standard Deviation, Binding Assay

    ELISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a ) Single-dilution ELISA to evaluate the presence of RBD-reactive human IgG in pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:50 dilution. Samples were evaluated in biological duplicates and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( b ) Dilution series ELISA was performed to quantitate RBD-reactive human IgG in plasma. Samples were evaluated in biological duplicates. Dashed curves represent fit lines from a four-parameter logistic regression applied over each series. ( c ) Data from ( b ) plotted as area-under-the-curve (AUC).
    Figure Legend Snippet: ELISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a ) Single-dilution ELISA to evaluate the presence of RBD-reactive human IgG in pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:50 dilution. Samples were evaluated in biological duplicates and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( b ) Dilution series ELISA was performed to quantitate RBD-reactive human IgG in plasma. Samples were evaluated in biological duplicates. Dashed curves represent fit lines from a four-parameter logistic regression applied over each series. ( c ) Data from ( b ) plotted as area-under-the-curve (AUC).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Standard Deviation

    BLI-ISA evaluation of plasma antibodies to SARS-CoV-2 prefusion Spike and plasma IgA to SARS-CoV-2 spike RBD. ( a ) Single-dilution BLI-ISA to evaluate the presence of prefusion Spike-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples. The Total Antibody Binding signal (left) is measured when prefusion Spike-His-loaded HIS1K biosensors are dipped into plasma samples. The Detection signal (right) is measured when prefusion Spike-His-loaded HIS1K biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the samples. The Total Antibody Binding signal (left) is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. The Detection signal (right) is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgA. The SP7 dot is colored pink to indicate that this sample had a negative signal (value in parentheses) in the Detection step. All assays were performed with plasma at a 1:8 dilution. Dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean.
    Figure Legend Snippet: BLI-ISA evaluation of plasma antibodies to SARS-CoV-2 prefusion Spike and plasma IgA to SARS-CoV-2 spike RBD. ( a ) Single-dilution BLI-ISA to evaluate the presence of prefusion Spike-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples. The Total Antibody Binding signal (left) is measured when prefusion Spike-His-loaded HIS1K biosensors are dipped into plasma samples. The Detection signal (right) is measured when prefusion Spike-His-loaded HIS1K biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the samples. The Total Antibody Binding signal (left) is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. The Detection signal (right) is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgA. The SP7 dot is colored pink to indicate that this sample had a negative signal (value in parentheses) in the Detection step. All assays were performed with plasma at a 1:8 dilution. Dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean.

    Techniques Used: Binding Assay, Standard Deviation

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    Next-Generation Sequencing:

    Article Title: “Acute Respiratory Distress and Cytokine Storm in Aged, SARS-CoV-2 Infected African Green Monkeys, but not in Rhesus Macaques”
    Article Snippet: .. Tissues were blocked with 10% normal goat serum (NGS) for 40 minutes, followed by a 60-minute incubation with the primary antibodies (SARS-CoV-2 nucleoprotein, mouse IgG1 (Sino Biological, cat#40143-MM08); ACE2, rabbit polyclonal (Millipore, cat# HPA000288); Iba-1, rabbit polyclonal (Wako, cat# 019-19741); or pancytokeratin, rabbit polyclonal (Dako, cat#Z0622)) diluted in NGS at a concentration of 1:200 and 1:100, respectively). ..

    Blocking Assay:

    Article Title: A novel biparatopic antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy
    Article Snippet: .. Cell based blocking testGradient diluted antibodies or antibody-ACE2 fusion proteins were first pre-incubated with 0.1nM S1-mFc (SinoBiological) at 37 °C overnight, followed by incubation with CHO cells stably expressing ACE2 protein (CHO-ACE2) for 1h at RT. .. The cells were washed and subsequently incubated with APC-labeled goat anti-mouse IgG (ebioscience) at 4 °C for 30 min. APC fluorescence signals were determined using a Beckman flow cytometer and results were analyzed using GraphPad7 software.

    Article Title: A novel biparatopic hybrid antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy
    Article Snippet: .. Cell-based blocking testGradient diluted antibodies or antibody-ACE2 fusion proteins were first pre-incubated with 0.1 nM S1-mFc (Sino Biological) at 37°C overnight, followed by incubation with CHO cells stably expressing ACE2 protein (CHO-ACE2) for 1 h at room temperature. .. The cells were washed and subsequently incubated with allophycocyanin (APC)-labeled goat anti-mouse IgG (Biolegend, catalog#405308) at 4°C for 30 min. APC fluorescence signals were determined using a Beckman flow cytometer and results were analyzed using GraphPad7 software.

    Concentration Assay:

    Article Title: “Acute Respiratory Distress and Cytokine Storm in Aged, SARS-CoV-2 Infected African Green Monkeys, but not in Rhesus Macaques”
    Article Snippet: .. Tissues were blocked with 10% normal goat serum (NGS) for 40 minutes, followed by a 60-minute incubation with the primary antibodies (SARS-CoV-2 nucleoprotein, mouse IgG1 (Sino Biological, cat#40143-MM08); ACE2, rabbit polyclonal (Millipore, cat# HPA000288); Iba-1, rabbit polyclonal (Wako, cat# 019-19741); or pancytokeratin, rabbit polyclonal (Dako, cat#Z0622)) diluted in NGS at a concentration of 1:200 and 1:100, respectively). ..

    Incubation:

    Article Title: A novel biparatopic antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy
    Article Snippet: .. Cell based blocking testGradient diluted antibodies or antibody-ACE2 fusion proteins were first pre-incubated with 0.1nM S1-mFc (SinoBiological) at 37 °C overnight, followed by incubation with CHO cells stably expressing ACE2 protein (CHO-ACE2) for 1h at RT. .. The cells were washed and subsequently incubated with APC-labeled goat anti-mouse IgG (ebioscience) at 4 °C for 30 min. APC fluorescence signals were determined using a Beckman flow cytometer and results were analyzed using GraphPad7 software.

    Article Title: “Acute Respiratory Distress and Cytokine Storm in Aged, SARS-CoV-2 Infected African Green Monkeys, but not in Rhesus Macaques”
    Article Snippet: .. Tissues were blocked with 10% normal goat serum (NGS) for 40 minutes, followed by a 60-minute incubation with the primary antibodies (SARS-CoV-2 nucleoprotein, mouse IgG1 (Sino Biological, cat#40143-MM08); ACE2, rabbit polyclonal (Millipore, cat# HPA000288); Iba-1, rabbit polyclonal (Wako, cat# 019-19741); or pancytokeratin, rabbit polyclonal (Dako, cat#Z0622)) diluted in NGS at a concentration of 1:200 and 1:100, respectively). ..

    Article Title: A novel biparatopic hybrid antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy
    Article Snippet: .. Cell-based blocking testGradient diluted antibodies or antibody-ACE2 fusion proteins were first pre-incubated with 0.1 nM S1-mFc (Sino Biological) at 37°C overnight, followed by incubation with CHO cells stably expressing ACE2 protein (CHO-ACE2) for 1 h at room temperature. .. The cells were washed and subsequently incubated with allophycocyanin (APC)-labeled goat anti-mouse IgG (Biolegend, catalog#405308) at 4°C for 30 min. APC fluorescence signals were determined using a Beckman flow cytometer and results were analyzed using GraphPad7 software.

    Infection:

    Article Title: Peptide Antidotes to SARS-CoV-2 (COVID-19)
    Article Snippet: .. A neutralizing monoclonal IgG antibody against the SARS-CoV-2 spike glycoprotein (CR3022, antibodies-online), ACE2 (Sino Biological), receptor-binding domain (RBD) of spike glycoprotein (Sino Biological), and SARS-BLOCK™ peptides (Ligandal) were used as inhibitors of infection. .. Infection was quantitated via bioluminescence, and toxicity was characterized via a trypan blue absorbance assay utilizing a Synergy™ H1 BioTek spectrophotometer 60h following viral transduction.

    Cell Culture:

    Article Title: Peptide Antidotes to SARS-CoV-2 (COVID-19)
    Article Snippet: .. LF performed cell culture and lentiviral transduction of HEK-ACE2 cells in the presence of peptides, antibodies, RBD, and ACE2. .. AW performed spectrophotometry and trypan blue assays on the transduced cells.

    Expressing:

    Article Title: A novel biparatopic antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy
    Article Snippet: .. Cell based blocking testGradient diluted antibodies or antibody-ACE2 fusion proteins were first pre-incubated with 0.1nM S1-mFc (SinoBiological) at 37 °C overnight, followed by incubation with CHO cells stably expressing ACE2 protein (CHO-ACE2) for 1h at RT. .. The cells were washed and subsequently incubated with APC-labeled goat anti-mouse IgG (ebioscience) at 4 °C for 30 min. APC fluorescence signals were determined using a Beckman flow cytometer and results were analyzed using GraphPad7 software.

    Article Title: A novel biparatopic hybrid antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy
    Article Snippet: .. Cell-based blocking testGradient diluted antibodies or antibody-ACE2 fusion proteins were first pre-incubated with 0.1 nM S1-mFc (Sino Biological) at 37°C overnight, followed by incubation with CHO cells stably expressing ACE2 protein (CHO-ACE2) for 1 h at room temperature. .. The cells were washed and subsequently incubated with allophycocyanin (APC)-labeled goat anti-mouse IgG (Biolegend, catalog#405308) at 4°C for 30 min. APC fluorescence signals were determined using a Beckman flow cytometer and results were analyzed using GraphPad7 software.

    Binding Assay:

    Article Title: Rapid High-Yield Production of Functional SARS-CoV-2 Receptor Binding Domain by Viral and Non-Viral Transient Expression for Pre-Clinical Evaluation
    Article Snippet: .. These experiments were run simultaneously, thus the anti-ACE2 mAb served as positive control of binding for all the analysis conducted. .. The standard S1 subunit used as reference in the study corresponds to SARS-Cov-2 DNA sequence encoding YP_009724390.1, consisting of 681 amino acids (76.5 kDa) with a polyhistidine tag at the C-terminus (Cat: 40591-V08H; Sino Biological).

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    Sino Biological sars cov 2 2019 ncov nucleocapsid antibody rabbit pab
    ILRUN supresses <t>SARS-CoV-2</t> infection and down-regulates host genes essential for SARS-CoV-2 entry. (A) Transcription profile of SARS-CoV-2 in Caco-2 cells transfected with 40 nM siNEG or siILRUN for 72 h at 6 h and 24 h post infection. (B) SARS-CoV-2 titres of supernatants from Caco-2 cells infected with SARS-CoV-2 (24 h, MOI 0.3) post-transfection with siRNAs (40 nM, 72 h) *p
    Sars Cov 2 2019 Ncov Nucleocapsid Antibody Rabbit Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Sino Biological sars cov 2 s1 s2
    The levels of <t>SARS-CoV-2-reactive</t> to S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic. The levels of a S1 + S2- and b nucleocapsid-reactive secretory IgM (SIgM)/IgM, IgG, and secretory IgA (SIgA)/IgA in human milk. Values are mean ± SD, n = 41 for women. Asterisks show statistically significant differences between variables (*** p
    Sars Cov 2 S1 S2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Sino Biological sars cov 2 2019 ncov spike antibody rabbit pab
    Inhibitory activity of HP-OVA against <t>SARS-CoV-2</t> S-mediated cell-cell fusion. Images were captured at 12 h after treatment with HP-OVA or OVA on SARS-CoV-2 S protein-mediated cell-cell fusion. The syncytia of Vero E6 cells (A) or Huh 7 cells (B) and HEK293T cells with SARS-CoV-2 overexpression are marked in the pictures. Representative results from three fields were selected randomly from each sample with scale bars of 50 μm (C, D) The number of syncytia was counted under an inverted fluorescence microscope, and the percentage of inhibition was calculated as described in the Methods. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p
    Sars Cov 2 2019 Ncov Spike Antibody Rabbit Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ILRUN supresses SARS-CoV-2 infection and down-regulates host genes essential for SARS-CoV-2 entry. (A) Transcription profile of SARS-CoV-2 in Caco-2 cells transfected with 40 nM siNEG or siILRUN for 72 h at 6 h and 24 h post infection. (B) SARS-CoV-2 titres of supernatants from Caco-2 cells infected with SARS-CoV-2 (24 h, MOI 0.3) post-transfection with siRNAs (40 nM, 72 h) *p

    Journal: bioRxiv

    Article Title: ILRUN downregulates ACE2 expression and blocks infection of human cells by SARS-CoV-2

    doi: 10.1101/2020.11.13.381343

    Figure Lengend Snippet: ILRUN supresses SARS-CoV-2 infection and down-regulates host genes essential for SARS-CoV-2 entry. (A) Transcription profile of SARS-CoV-2 in Caco-2 cells transfected with 40 nM siNEG or siILRUN for 72 h at 6 h and 24 h post infection. (B) SARS-CoV-2 titres of supernatants from Caco-2 cells infected with SARS-CoV-2 (24 h, MOI 0.3) post-transfection with siRNAs (40 nM, 72 h) *p

    Article Snippet: Immunofluorescence and quantification of relative antigen stainingCaco-2 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008).

    Techniques: Infection, Transfection

    Validation of ILRUN function and SARS-CoV-2 infection in Caco-2 cells. (A) ILRUN mRNA levels (2 −ΔΔCt relative to GAPDH ) in Caco-2 cells transfected with siRNAs (40 nM, 72 h) targeting ILRUN or a nontargeting control (siNEG). **p

    Journal: bioRxiv

    Article Title: ILRUN downregulates ACE2 expression and blocks infection of human cells by SARS-CoV-2

    doi: 10.1101/2020.11.13.381343

    Figure Lengend Snippet: Validation of ILRUN function and SARS-CoV-2 infection in Caco-2 cells. (A) ILRUN mRNA levels (2 −ΔΔCt relative to GAPDH ) in Caco-2 cells transfected with siRNAs (40 nM, 72 h) targeting ILRUN or a nontargeting control (siNEG). **p

    Article Snippet: Immunofluorescence and quantification of relative antigen stainingCaco-2 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008).

    Techniques: Infection, Transfection

    The levels of SARS-CoV-2-reactive to S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic. The levels of a S1 + S2- and b nucleocapsid-reactive secretory IgM (SIgM)/IgM, IgG, and secretory IgA (SIgA)/IgA in human milk. Values are mean ± SD, n = 41 for women. Asterisks show statistically significant differences between variables (*** p

    Journal: Journal of Perinatology

    Article Title: Difference in levels of SARS-CoV-2 S1 and S2 subunits- and nucleocapsid protein-reactive SIgM/IgM, IgG and SIgA/IgA antibodies in human milk

    doi: 10.1038/s41372-020-00805-w

    Figure Lengend Snippet: The levels of SARS-CoV-2-reactive to S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic. The levels of a S1 + S2- and b nucleocapsid-reactive secretory IgM (SIgM)/IgM, IgG, and secretory IgA (SIgA)/IgA in human milk. Values are mean ± SD, n = 41 for women. Asterisks show statistically significant differences between variables (*** p

    Article Snippet: SARS-CoV-2 S1 + S2- and nucleocapsid-reactive antibodies The levels of SARS-CoV-2 S1 + S2 and nucleocapsid-reactive SIgM/IgM, IgG and SIgA/IgA were determined using ELISAs that were adapted from our previous publications [ , – ].

    Techniques:

    Regression linear between antibodies reactive to SARS-CoV-2 S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic in 41 women. a Positive correlation of S1 + S2-reactive secretory IgA (SIgA)/IgA and S1 + S2-reactive secretory IgM (SIgM)/IgM. b Positive correlation between S1 + S2-reactive SIgA/IgA and nucleocapsid-reactive SIgA/IgA. c Positive correlation between nucleocapsid-reactive SIgA/IgA and SIgM/IgM. d Positive correlation between nucleocapsid-reactive SIgM/IgM and S1 + S2-reactive SIgM/IgM. Pearson correlation coefficients ( r ) were determined when p

    Journal: Journal of Perinatology

    Article Title: Difference in levels of SARS-CoV-2 S1 and S2 subunits- and nucleocapsid protein-reactive SIgM/IgM, IgG and SIgA/IgA antibodies in human milk

    doi: 10.1038/s41372-020-00805-w

    Figure Lengend Snippet: Regression linear between antibodies reactive to SARS-CoV-2 S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic in 41 women. a Positive correlation of S1 + S2-reactive secretory IgA (SIgA)/IgA and S1 + S2-reactive secretory IgM (SIgM)/IgM. b Positive correlation between S1 + S2-reactive SIgA/IgA and nucleocapsid-reactive SIgA/IgA. c Positive correlation between nucleocapsid-reactive SIgA/IgA and SIgM/IgM. d Positive correlation between nucleocapsid-reactive SIgM/IgM and S1 + S2-reactive SIgM/IgM. Pearson correlation coefficients ( r ) were determined when p

    Article Snippet: SARS-CoV-2 S1 + S2- and nucleocapsid-reactive antibodies The levels of SARS-CoV-2 S1 + S2 and nucleocapsid-reactive SIgM/IgM, IgG and SIgA/IgA were determined using ELISAs that were adapted from our previous publications [ , – ].

    Techniques:

    The levels of antibodies reactive to SARS-CoV-2 S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic (2020-HM) and 2 years prior this pandemic (2018-HM). Levels of S1 + S2-reactive a secretory IgM (SIgM)/IgM, b IgG and c secretory IgA (SIgA)/IgA in 2020-HM and 2018-HM. Levels of nucleocapsid-reactive d SIgM/IgM, e IgG and f SIgA/IgA in 2020-HM and 2018-HM. Values are mean ± SD, n = 41 for 2020-HM and n = 16 for 2018-HM. Asterisks show statistically significant differences between variables (* p

    Journal: Journal of Perinatology

    Article Title: Difference in levels of SARS-CoV-2 S1 and S2 subunits- and nucleocapsid protein-reactive SIgM/IgM, IgG and SIgA/IgA antibodies in human milk

    doi: 10.1038/s41372-020-00805-w

    Figure Lengend Snippet: The levels of antibodies reactive to SARS-CoV-2 S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic (2020-HM) and 2 years prior this pandemic (2018-HM). Levels of S1 + S2-reactive a secretory IgM (SIgM)/IgM, b IgG and c secretory IgA (SIgA)/IgA in 2020-HM and 2018-HM. Levels of nucleocapsid-reactive d SIgM/IgM, e IgG and f SIgA/IgA in 2020-HM and 2018-HM. Values are mean ± SD, n = 41 for 2020-HM and n = 16 for 2018-HM. Asterisks show statistically significant differences between variables (* p

    Article Snippet: SARS-CoV-2 S1 + S2- and nucleocapsid-reactive antibodies The levels of SARS-CoV-2 S1 + S2 and nucleocapsid-reactive SIgM/IgM, IgG and SIgA/IgA were determined using ELISAs that were adapted from our previous publications [ , – ].

    Techniques:

    Percentage of detected antibodies reactive to S1 and S2 subunits (S1 + S2), and nucleocapsid from SARS-CoV-2 in human milk collected during the COVID-19 pandemic. S1+S2-reactive a secretory IgM (SIgM)/IgM, b IgG and c secretory IgA (SIgA)/IgA in human milk from 41 women. Nucleocapsid-reactive d SIgM/IgM, e IgG and f SIgA/IgA. Milk expression period was from 30/02/20 to 03/04/20 in the United States.

    Journal: Journal of Perinatology

    Article Title: Difference in levels of SARS-CoV-2 S1 and S2 subunits- and nucleocapsid protein-reactive SIgM/IgM, IgG and SIgA/IgA antibodies in human milk

    doi: 10.1038/s41372-020-00805-w

    Figure Lengend Snippet: Percentage of detected antibodies reactive to S1 and S2 subunits (S1 + S2), and nucleocapsid from SARS-CoV-2 in human milk collected during the COVID-19 pandemic. S1+S2-reactive a secretory IgM (SIgM)/IgM, b IgG and c secretory IgA (SIgA)/IgA in human milk from 41 women. Nucleocapsid-reactive d SIgM/IgM, e IgG and f SIgA/IgA. Milk expression period was from 30/02/20 to 03/04/20 in the United States.

    Article Snippet: SARS-CoV-2 S1 + S2- and nucleocapsid-reactive antibodies The levels of SARS-CoV-2 S1 + S2 and nucleocapsid-reactive SIgM/IgM, IgG and SIgA/IgA were determined using ELISAs that were adapted from our previous publications [ , – ].

    Techniques: Expressing

    Inhibitory activity of HP-OVA against SARS-CoV-2 S-mediated cell-cell fusion. Images were captured at 12 h after treatment with HP-OVA or OVA on SARS-CoV-2 S protein-mediated cell-cell fusion. The syncytia of Vero E6 cells (A) or Huh 7 cells (B) and HEK293T cells with SARS-CoV-2 overexpression are marked in the pictures. Representative results from three fields were selected randomly from each sample with scale bars of 50 μm (C, D) The number of syncytia was counted under an inverted fluorescence microscope, and the percentage of inhibition was calculated as described in the Methods. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Journal: Frontiers in Pharmacology

    Article Title: 3-Hydroxyphthalic Anhydride-Modified Chicken Ovalbumin as a Potential Candidate Inhibits SARS-CoV-2 Infection by Disrupting the Interaction of Spike Protein With Host ACE2 Receptor

    doi: 10.3389/fphar.2020.603830

    Figure Lengend Snippet: Inhibitory activity of HP-OVA against SARS-CoV-2 S-mediated cell-cell fusion. Images were captured at 12 h after treatment with HP-OVA or OVA on SARS-CoV-2 S protein-mediated cell-cell fusion. The syncytia of Vero E6 cells (A) or Huh 7 cells (B) and HEK293T cells with SARS-CoV-2 overexpression are marked in the pictures. Representative results from three fields were selected randomly from each sample with scale bars of 50 μm (C, D) The number of syncytia was counted under an inverted fluorescence microscope, and the percentage of inhibition was calculated as described in the Methods. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA)ELISA was performed to identify the interaction of HP-OVA and the SARS-CoV-2 S protein (RBD) or ACE2 protein.

    Techniques: Activity Assay, Over Expression, Fluorescence, Microscopy, Inhibition

    Inhibition of HP-OVA on the infection with SARS-CoV-2 PsV and SARS-CoV PsV. Antiviral activity of HP-OVA against SARS-CoV-2 S PsV infection in 293T/ACE2 (A) or Vero E6 (B) target cells. Inhibition of single-round infection of SARS-CoV S PsV in 293T/ACE2 (C) and Vero E6 (D) cells. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Journal: Frontiers in Pharmacology

    Article Title: 3-Hydroxyphthalic Anhydride-Modified Chicken Ovalbumin as a Potential Candidate Inhibits SARS-CoV-2 Infection by Disrupting the Interaction of Spike Protein With Host ACE2 Receptor

    doi: 10.3389/fphar.2020.603830

    Figure Lengend Snippet: Inhibition of HP-OVA on the infection with SARS-CoV-2 PsV and SARS-CoV PsV. Antiviral activity of HP-OVA against SARS-CoV-2 S PsV infection in 293T/ACE2 (A) or Vero E6 (B) target cells. Inhibition of single-round infection of SARS-CoV S PsV in 293T/ACE2 (C) and Vero E6 (D) cells. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA)ELISA was performed to identify the interaction of HP-OVA and the SARS-CoV-2 S protein (RBD) or ACE2 protein.

    Techniques: Inhibition, Infection, Activity Assay

    HP-OVA binding to both SARS-CoV-2 S and ACE2 protein. Analysis of the expression of SARS-CoV-2 S (A) and ACE2 (B) in HEK-293T cells by western blot. The binding of HP-OVA to cells expressing SARS-CoV-2 S (C) or ACE2 (D) was assessed by flow cytometry. A representative flow histogram and quantification of the binding of HP-OVA to cells expressing SARS-CoV-2 S (E) or ACE2 (F) were shown. Data are presented as the mean ± SD (* p

    Journal: Frontiers in Pharmacology

    Article Title: 3-Hydroxyphthalic Anhydride-Modified Chicken Ovalbumin as a Potential Candidate Inhibits SARS-CoV-2 Infection by Disrupting the Interaction of Spike Protein With Host ACE2 Receptor

    doi: 10.3389/fphar.2020.603830

    Figure Lengend Snippet: HP-OVA binding to both SARS-CoV-2 S and ACE2 protein. Analysis of the expression of SARS-CoV-2 S (A) and ACE2 (B) in HEK-293T cells by western blot. The binding of HP-OVA to cells expressing SARS-CoV-2 S (C) or ACE2 (D) was assessed by flow cytometry. A representative flow histogram and quantification of the binding of HP-OVA to cells expressing SARS-CoV-2 S (E) or ACE2 (F) were shown. Data are presented as the mean ± SD (* p

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA)ELISA was performed to identify the interaction of HP-OVA and the SARS-CoV-2 S protein (RBD) or ACE2 protein.

    Techniques: Binding Assay, Expressing, Western Blot, Flow Cytometry

    The interaction of HP-OVA with SARS-CoV-2 S and ACE2. The binding of OVA to SARS-CoV-2 spike (RBD), S2 and BSA protein was assessed by ELISA (A) . The binding of HP-OVA to SARS-CoV-2 spike (RBD), S2 and a negative control BSA protein was assessed by ELISA (B) . The binding ability of HP-OVA to ACE2 protein was assessed by ELISA (C) . Inhibition of the interaction between spike (RBD) and ACE2 proteins by HP-OVA, as determined by a competitive inhibition ELISA (D) . Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Journal: Frontiers in Pharmacology

    Article Title: 3-Hydroxyphthalic Anhydride-Modified Chicken Ovalbumin as a Potential Candidate Inhibits SARS-CoV-2 Infection by Disrupting the Interaction of Spike Protein With Host ACE2 Receptor

    doi: 10.3389/fphar.2020.603830

    Figure Lengend Snippet: The interaction of HP-OVA with SARS-CoV-2 S and ACE2. The binding of OVA to SARS-CoV-2 spike (RBD), S2 and BSA protein was assessed by ELISA (A) . The binding of HP-OVA to SARS-CoV-2 spike (RBD), S2 and a negative control BSA protein was assessed by ELISA (B) . The binding ability of HP-OVA to ACE2 protein was assessed by ELISA (C) . Inhibition of the interaction between spike (RBD) and ACE2 proteins by HP-OVA, as determined by a competitive inhibition ELISA (D) . Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA)ELISA was performed to identify the interaction of HP-OVA and the SARS-CoV-2 S protein (RBD) or ACE2 protein.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Inhibition

    Schematic representation of the molecular mechanisms of HP-OVA against SARS-CoV-2 infection. HP-OVA binds to both the S protein of SARS-CoV-2 and host angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV-2, and disrupts the S protein-ACE2 interaction, thereby exhibiting inhibitory activity against SARS-CoV-2 infection.

    Journal: Frontiers in Pharmacology

    Article Title: 3-Hydroxyphthalic Anhydride-Modified Chicken Ovalbumin as a Potential Candidate Inhibits SARS-CoV-2 Infection by Disrupting the Interaction of Spike Protein With Host ACE2 Receptor

    doi: 10.3389/fphar.2020.603830

    Figure Lengend Snippet: Schematic representation of the molecular mechanisms of HP-OVA against SARS-CoV-2 infection. HP-OVA binds to both the S protein of SARS-CoV-2 and host angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV-2, and disrupts the S protein-ACE2 interaction, thereby exhibiting inhibitory activity against SARS-CoV-2 infection.

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA)ELISA was performed to identify the interaction of HP-OVA and the SARS-CoV-2 S protein (RBD) or ACE2 protein.

    Techniques: Infection, Functional Assay, Activity Assay

    A model for structural rearrangements of SARS-Cov-2 S protein. (A) Structural changes independent of a target cell. We suggest that both the prefusion and postfusion spikes are present on the surface of mature virion and the ratio between them may vary (diagram of virion). The postfusion spikes on the virion are formed by S2 after S1 dissociates in the absence of ACE2. (B) ACE2-dependent structural rearrangements. Structural transition from the prefusion to postfusion conformation inducing membrane fusion likely proceeds stepwise as follows: 1) FPPR clamps down RBD through CTD1 in the prefusion S trimer, but it occasionally flips out of position and allows an RBD to sample the up conformation. 2) RBD binding to ACE2 creates a flexible FPPR that enables exposure of the S2’ cleavage site immediately upstream of the adjacent fusion peptide (FP). Cleavage at the S2’ site, and perhaps also the S1/S2 site, releases the structural constraints on the fusion peptide and initiates a cascade of refolding events in S2, probably accompanied by complete dissociation of S1. 3) Formation of the long central three-stranded coiled-coil and folding back of HR2. 4) Formation of the postfusion structure of S2 that brings the two membranes together, facilitating formation of a fusion pore and viral entry.

    Journal: bioRxiv

    Article Title: Distinct conformational states of SARS-CoV-2 spike protein

    doi: 10.1101/2020.05.16.099317

    Figure Lengend Snippet: A model for structural rearrangements of SARS-Cov-2 S protein. (A) Structural changes independent of a target cell. We suggest that both the prefusion and postfusion spikes are present on the surface of mature virion and the ratio between them may vary (diagram of virion). The postfusion spikes on the virion are formed by S2 after S1 dissociates in the absence of ACE2. (B) ACE2-dependent structural rearrangements. Structural transition from the prefusion to postfusion conformation inducing membrane fusion likely proceeds stepwise as follows: 1) FPPR clamps down RBD through CTD1 in the prefusion S trimer, but it occasionally flips out of position and allows an RBD to sample the up conformation. 2) RBD binding to ACE2 creates a flexible FPPR that enables exposure of the S2’ cleavage site immediately upstream of the adjacent fusion peptide (FP). Cleavage at the S2’ site, and perhaps also the S1/S2 site, releases the structural constraints on the fusion peptide and initiates a cascade of refolding events in S2, probably accompanied by complete dissociation of S1. 3) Formation of the long central three-stranded coiled-coil and folding back of HR2. 4) Formation of the postfusion structure of S2 that brings the two membranes together, facilitating formation of a fusion pore and viral entry.

    Article Snippet: Membranes were blocked with 5% skimmed milk in PBS for 1 hour and incubated either with anti-strep tag antibody (IBA Lifesciences) or anti-SARS-COV-2 polyclone antibody (Sino Biological Inc.) for another hour at room temperature.

    Techniques: Binding Assay

    Cryo-EM structure of the SARS-CoV-2 S2 in the postfusion conformation. (A) The structure of the S2 trimer was modeled based on a 3.3Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of the S2 trimer in the postfusion conformation shown in ribbon diagram. Various structural components in the color scheme shown in Fig. 1A include HR1, heptad repeat 1; CH, central helix region; CD, connector domain; and HR1, heptad repeat 2. The S2’ cleavage site is in a disordered loop between Ile770 and Thr912. A possible location of the S2 N-terminus (S1/S2 cleavage site) is also indicated. (C) A low resolution map showing the density pattern for 5 N-linked glycans, with almost equal spacing along the long axis.

    Journal: bioRxiv

    Article Title: Distinct conformational states of SARS-CoV-2 spike protein

    doi: 10.1101/2020.05.16.099317

    Figure Lengend Snippet: Cryo-EM structure of the SARS-CoV-2 S2 in the postfusion conformation. (A) The structure of the S2 trimer was modeled based on a 3.3Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of the S2 trimer in the postfusion conformation shown in ribbon diagram. Various structural components in the color scheme shown in Fig. 1A include HR1, heptad repeat 1; CH, central helix region; CD, connector domain; and HR1, heptad repeat 2. The S2’ cleavage site is in a disordered loop between Ile770 and Thr912. A possible location of the S2 N-terminus (S1/S2 cleavage site) is also indicated. (C) A low resolution map showing the density pattern for 5 N-linked glycans, with almost equal spacing along the long axis.

    Article Snippet: Membranes were blocked with 5% skimmed milk in PBS for 1 hour and incubated either with anti-strep tag antibody (IBA Lifesciences) or anti-SARS-COV-2 polyclone antibody (Sino Biological Inc.) for another hour at room temperature.

    Techniques:

    Selected new features of the SARS-CoV-2 prefusion S trimer. (A) N-terminal segment of S protein. The N-terminus is at residue Gln14 after cleavage of the signal peptide. Cys15 forms a disulfide bond with Cys136. We observed good density for the N-linked glycan at Asn17. (B) A segment immediately downstream of the fusion peptide, while disordered in the stabilized soluble S ectodomain trimer structure, forms a tightly packed structure, designated FPPR for the fusion peptide proximal region, abutting CTD1. The newly identified FPPR structure would clash with CTD1 in the RBD up conformation. Various domains are shown in the color scheme in Fig. 2B . The structure of the soluble S trimer with one RBD in the up conformation (PDB ID: 6vyb) is shown in gray. In the box, a close-up view of the FPPR with adjacent fusion peptide in both surface representation and stick model. (C) The SARS-CoV-2 prefusion S trimer, viewed along the threefold axis, is superposed on the structure of the stabilized soluble S ectodomain trimer in the closed conformation with all three RBDs in the down conformation (PDB ID: 6vxx). While the S2 region is well aligned, there is a significant shift (e.g. ∼10Å between two Ala123 residues) in S1. (D) Impact of the proline mutations introduced at residues 986 and 987 to stabilize the prefusion conformation. K986P mutation removes a salt bridge between Lys986 of one protomer and either Asp426 or Asp427 of another protomer in the trimer interface.

    Journal: bioRxiv

    Article Title: Distinct conformational states of SARS-CoV-2 spike protein

    doi: 10.1101/2020.05.16.099317

    Figure Lengend Snippet: Selected new features of the SARS-CoV-2 prefusion S trimer. (A) N-terminal segment of S protein. The N-terminus is at residue Gln14 after cleavage of the signal peptide. Cys15 forms a disulfide bond with Cys136. We observed good density for the N-linked glycan at Asn17. (B) A segment immediately downstream of the fusion peptide, while disordered in the stabilized soluble S ectodomain trimer structure, forms a tightly packed structure, designated FPPR for the fusion peptide proximal region, abutting CTD1. The newly identified FPPR structure would clash with CTD1 in the RBD up conformation. Various domains are shown in the color scheme in Fig. 2B . The structure of the soluble S trimer with one RBD in the up conformation (PDB ID: 6vyb) is shown in gray. In the box, a close-up view of the FPPR with adjacent fusion peptide in both surface representation and stick model. (C) The SARS-CoV-2 prefusion S trimer, viewed along the threefold axis, is superposed on the structure of the stabilized soluble S ectodomain trimer in the closed conformation with all three RBDs in the down conformation (PDB ID: 6vxx). While the S2 region is well aligned, there is a significant shift (e.g. ∼10Å between two Ala123 residues) in S1. (D) Impact of the proline mutations introduced at residues 986 and 987 to stabilize the prefusion conformation. K986P mutation removes a salt bridge between Lys986 of one protomer and either Asp426 or Asp427 of another protomer in the trimer interface.

    Article Snippet: Membranes were blocked with 5% skimmed milk in PBS for 1 hour and incubated either with anti-strep tag antibody (IBA Lifesciences) or anti-SARS-COV-2 polyclone antibody (Sino Biological Inc.) for another hour at room temperature.

    Techniques: Mutagenesis

    Preparation of a full-length SARS-CoV-2 spike protein. (A) Schematic representation of the expression construct of full-length SARS-CoV-2 spike (S) protein. Segments of S1 and S2 include: NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; S1/S2, S1/S2 cleavage site; S2’, S2’ cleavage site; FP, fusion peptide; FFPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane anchor; CT, cytoplasmic tail; and tree-like symbols for glycans. A strep-tag was fused to the C-terminus of S protein by a flexible linker. (B) The purified S protein was resolved by gel-filtration chromatography on a Superose 6 column in the presence of detergent NP-40. The molecular weight standards include thyoglobulin (670 kDa), γ-globulin (158 kDa) and ovalbumin (44 kDa). Three major peaks (peak I-III) contain the S protein. (C) Load sample and peak fractions from (B) were analyzed by Coomassie stained SDS-PAGE. Labeled bands were confirmed by western blot (S, S1 and S2) or protein sequencing (S2 and Cont; S and S1 bands did not gave any meaningful results probably due to a blocked N-terminus). Cont, copurified contaminating protein, identified as endoplasmic reticulum chaperone BiP precursor by N-terminal sequencing. Representative images and 2D averages by negative stain EM of three peak fractions are also shown. The box size of 2D averages is ∼510Å.

    Journal: bioRxiv

    Article Title: Distinct conformational states of SARS-CoV-2 spike protein

    doi: 10.1101/2020.05.16.099317

    Figure Lengend Snippet: Preparation of a full-length SARS-CoV-2 spike protein. (A) Schematic representation of the expression construct of full-length SARS-CoV-2 spike (S) protein. Segments of S1 and S2 include: NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; S1/S2, S1/S2 cleavage site; S2’, S2’ cleavage site; FP, fusion peptide; FFPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane anchor; CT, cytoplasmic tail; and tree-like symbols for glycans. A strep-tag was fused to the C-terminus of S protein by a flexible linker. (B) The purified S protein was resolved by gel-filtration chromatography on a Superose 6 column in the presence of detergent NP-40. The molecular weight standards include thyoglobulin (670 kDa), γ-globulin (158 kDa) and ovalbumin (44 kDa). Three major peaks (peak I-III) contain the S protein. (C) Load sample and peak fractions from (B) were analyzed by Coomassie stained SDS-PAGE. Labeled bands were confirmed by western blot (S, S1 and S2) or protein sequencing (S2 and Cont; S and S1 bands did not gave any meaningful results probably due to a blocked N-terminus). Cont, copurified contaminating protein, identified as endoplasmic reticulum chaperone BiP precursor by N-terminal sequencing. Representative images and 2D averages by negative stain EM of three peak fractions are also shown. The box size of 2D averages is ∼510Å.

    Article Snippet: Membranes were blocked with 5% skimmed milk in PBS for 1 hour and incubated either with anti-strep tag antibody (IBA Lifesciences) or anti-SARS-COV-2 polyclone antibody (Sino Biological Inc.) for another hour at room temperature.

    Techniques: Expressing, Construct, Binding Assay, Strep-tag, Purification, Filtration, Chromatography, Molecular Weight, Staining, SDS Page, Labeling, Western Blot, Sequencing

    Cryo-EM structure of the SARS-CoV-2 S protein in the prefusion conformation. (A) The structure of the S trimer was modeled based on a 3.1Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of S protein in the prefusion conformation shown in ribbon representation. Various structural components in the color scheme shown in Fig. 1A include NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; and CD, connector domain. N-terminus, S1/S2 cleavage site and S2’ cleavage site are indicated.

    Journal: bioRxiv

    Article Title: Distinct conformational states of SARS-CoV-2 spike protein

    doi: 10.1101/2020.05.16.099317

    Figure Lengend Snippet: Cryo-EM structure of the SARS-CoV-2 S protein in the prefusion conformation. (A) The structure of the S trimer was modeled based on a 3.1Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of S protein in the prefusion conformation shown in ribbon representation. Various structural components in the color scheme shown in Fig. 1A include NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; and CD, connector domain. N-terminus, S1/S2 cleavage site and S2’ cleavage site are indicated.

    Article Snippet: Membranes were blocked with 5% skimmed milk in PBS for 1 hour and incubated either with anti-strep tag antibody (IBA Lifesciences) or anti-SARS-COV-2 polyclone antibody (Sino Biological Inc.) for another hour at room temperature.

    Techniques: Binding Assay