Spike protein was purchased from Sino Biologicals. Mitoxantrone was purchased from Microsource (Cat #01503278) and stored by the NCATS compound management department. Banoxantrone was purchased from Sigma (Catalog number: SML1854). pcDNA3.1-SARS-CoV-2-Spike plasmid was obtained from BEI resource7 (link). pLV-Mcherry was obtained from Addgene (Catalog number: 36084). Hoechst 33342 staining solution and proteinase K were purchased from Thermo Fisher Scientific (Catalog number: 62249 and AM2546).
Spike protein
Manufactured by Sino Biological
Sourced in China, United States
The spike protein is a key component of the SARS-CoV-2 virus. It plays a crucial role in the virus's ability to infect host cells by binding to the ACE2 receptor. The spike protein is a complex structure that has been extensively studied for its potential use in vaccine development and research.
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Lab products found in correlation
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Multiskan fc, by Thermo Fisher Scientific (2 mentions)
Plv mcherry, by Addgene (1 mentions)
Hoechst 33342 staining solution, by Thermo Fisher Scientific (1 mentions)
Aluminum hydroxide gel adjuvant, by InvivoGen (1 mentions)
Anti mouse iga, by Fortis Life Sciences (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Hrp conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Elisa plate, by Corning (1 mentions)
Tcs sp8 microscope, by Leica (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Ab97240, by Abcam (1 mentions)
Ab97245, by Abcam (1 mentions)
Ab6789, by Abcam (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Eia ria plate, by Corning (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Cr3022, by Absolute Antibody (1 mentions)
11 protocols using spike protein
1
SARS-CoV-2 Spike Protein Assay
2
SARS-CoV-2 Spike Protein Vaccine in Mice
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For each group, female Balb/c mice (6–8 weeks of age, n = 4) were immunized 3 times at day 0, 7 and 21 intramuscularly. Spike protein (Sino Biological, Beijing, China) was dissolved in PBS at the final concentration of 0.1 μg/μl. Alum-3M-052 was a mixture that contained 50 μl 2% aluminum hydroxide gel adjuvant (InvivoGen, France) and 2 μg 3M-052 (MedChemExpress, NJ, USA) in 50 ul PBS. For vaccine test group, 50 μl Spike protein solution and 50 μl Alum-3M-052 were mixed at 150 rpm for 30 min by a shaker (Kylin-Bell, Jiangsu, China) at 4°C. For vaccine control group, 50 μl Spike protein solution and 50 μl 2% aluminum hydroxide gel adjuvant were mixed at 150 rpm for 30 min by a shaker at 4°C. For negative control group, 50 μl PBS and 50 μl 2% aluminum hydroxide gel adjuvant were mixed at 150 rpm for 30 min by a shaker at 4°C. The blood samples were collected 1 week following each immunization.
3
Quantifying SARS-CoV-2 Spike Protein Adsorption
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Anti-S-protein antibody titers in serum or other biological fluids were determined using ELISA. Briefly, we coated 1 μg/ml spike protein (Sino Biological, PA, USA) onto ELISA plates (Corning, NY, USA) in PBS overnight at 4 °C or 2 hours at 37 °C. The plate was blocked with PBS +1 % BSA (Fisher Scientific, PA, USA) + 0.1 % Tween20TM (Sigma-Aldrich, MD, USA) for 2 hours at room temperature. After washing, we added the samples at different dilutions. We detected the antibodies by HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, 1 in 5,000; PA, USA), anti-mouse IgA (Bethyl Laboratories, 1 in 10,000; TX, USA), and detection antibody against mouse IgA (1 in 250) from the mouse total IgA ELISA kit from Invitrogen (CA, USA). The positive control (anti-S IgG) was obtained from Abeomics (CA, USA).
To estimate the fraction of the protein adsorbed onto the liposomes, trimeric S-protein (10 μl, 1 μg /μl) was gently mixed with NanoSTING (20 μg), and incubated at room temperature for 10 min. The mixture was centrifuged at 20,000 x g for 40 min, and the pellet was resuspend and washed in 25 μl PBS. All supernatant were collected and combined, and then the total volume was measured.
The total amount of S-protein, the fraction of S-protein sedimented on the NanoSTING, and the protein in the supernatant fraction were evaluated using a quantitative S-protein ELISA.
To estimate the fraction of the protein adsorbed onto the liposomes, trimeric S-protein (10 μl, 1 μg /μl) was gently mixed with NanoSTING (20 μg), and incubated at room temperature for 10 min. The mixture was centrifuged at 20,000 x g for 40 min, and the pellet was resuspend and washed in 25 μl PBS. All supernatant were collected and combined, and then the total volume was measured.
The total amount of S-protein, the fraction of S-protein sedimented on the NanoSTING, and the protein in the supernatant fraction were evaluated using a quantitative S-protein ELISA.
4
Spike Protein and Pseudovirus Binding Assay
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HEK293T, PANC02, and Huh7 cells (5 × 104) were seeded onto cover glass in 6-well plates. After 24 h, the cell cultures were replenished with DMEM and incubated with Spike protein (Sinobiological, Beijing, China), pseudovirus (Sinobiological, Beijing, China) or inactivated virus in a 5% CO2 incubator for 30 min at 37°C. The cells were fixed with 4% paraformaldehyde and subsequently blocked and permeabilized with phosphate-buffered saline with Tween-20 (PBST) containing 1% bovine serum albumin (BSA). Since the Spike protein and pseudovirus carried a His-tag, we incubated the cultures with an anti-His primary antibody overnight. The inactivated virus was incubated overnight with a Spike-neutralizing antibody. Alexa Fluor-488-conjugated streptavidin (Boster, Beijing, China) was added, and the cultures were incubated for 1 h. Then, the samples were washed with PBST, and Hoechst 33258 (Thermo Fisher Scientific, Waltham, MA, United States) was added to stain the nuclei. The slides were analysed by confocal microscopy using a TCS SP8 microscope (Leica, Germany).
5
SARS-CoV-2 Spike Protein Antibody ELISA
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ELISAs were designed to quantify SARS-CoV-2 Spike protein-reactive total IgG, IgG2a, and IgG1, as well as SARS-CoV Spike protein-reactive total IgG. Recombinant SARS-CoV-2, SARS-CoV, or B.1.351 Spike proteins (Sino Biological) were diluted to 200 ng/mL in 50 mM sodium carbonate buffer (pH 9.6), added to 96-well EIA/RIA plates (Corning) at 100 μL/well, and incubated overnight at 4°C. The plates were washed with PBS containing 0.5% Tween-20 (PBST) and blocked with 5% bovine serum albumin (BSA) in PBS for 30 min at 37°C. Mouse serum samples were serially diluted 5-fold in PBST containing 1% BSA and 100 μL was added to each well and incubated for 2 h at 37°C. The plates were washed three times with PBST and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Abcam, ab6789, 1:10,000), goat anti-mouse IgG1 (Abcam, ab97240, 1:10,000), or goat anti-mouse IgG2a (Abcam, ab97245, 1:10,000) for 1 h. The plates were washed with PBST, color was developed by addition of 3′,5,5′-tetramethylbenzidine (TMB) substrate, and the reaction was stopped by addition of 2 M HCl. The absorbance (optical density, OD) at 450 nm was measured using a microplate reader (BioTeK). The endpoint dilution titer was defined as the highest serum dilution giving an OD >2-fold the background OD of control wells consisting of diluted serum.
6
SARS-CoV-2 Antibody Detection Assay
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Binding plates (Costar) were coated with 100 ng/well Spike protein or Nucleocapsid protein (SinoBiological) at 4 °C overnight. The plates were blocked with 4% bovine serum albumin (BSA) for 90 min. After blocking, 50 μL of serum dilution was added to each well and incubated at 37 °C for 1 h. The plates were washed with PBS containing 0.05% Tween-20. Enzyme-labeled goat anti-human IgG-Fc or enzyme-labeled goat anti-human IgM-Fc was added as the secondary antibody. After incubation for 45 min, the plates were washed, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added, and then 100 μL of 2 mol/L H2SO4 was added to stop the reaction. The microplates were read using a microplate reader (Multiskan FC, Thermo Scientific) at 450 nm. The mean HC area under the curve (AUC) value + 3SD was used as the cutoff value to determine positive serum samples [6 (link)].
7
Evaluating SARS-CoV-2 Antibody Responses
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Female mice, 6–8 weeks old C57Bl were obtained from Janevier, Sollentuna, Sweden.
Human endogenous adjuvant L3 (anionic) and a derivative of the N3 (cationic) lipid formulations were obtained from Merck (Darmstadt, Germany). Spike protein and the recombinant NC protein were purchased from SinoBiological (SCG, Toronto ON, Canada/Sinobiologicals, Eschborn, Germany). For cells culture, DMEM plus 2 mM L-glutamax and Na-pyruvate was used. penicillin-streptomycine and a 10% inactivated bovine serum albumin (BSA) were added to the medium. The positive control used was obtained from SARS-CoV-2 infected individuals. The negative control serum was pre-infection serum. The medium, penicillin-streptomycin, BSA and PFA were purchased from Sigma-Aldrich/Merck (Solna, Sweden).
Human endogenous adjuvant L3 (anionic) and a derivative of the N3 (cationic) lipid formulations were obtained from Merck (Darmstadt, Germany). Spike protein and the recombinant NC protein were purchased from SinoBiological (SCG, Toronto ON, Canada/Sinobiologicals, Eschborn, Germany). For cells culture, DMEM plus 2 mM L-glutamax and Na-pyruvate was used. penicillin-streptomycine and a 10% inactivated bovine serum albumin (BSA) were added to the medium. The positive control used was obtained from SARS-CoV-2 infected individuals. The negative control serum was pre-infection serum. The medium, penicillin-streptomycin, BSA and PFA were purchased from Sigma-Aldrich/Merck (Solna, Sweden).
8
SARS-CoV-2 Spike and RBD ELISA Assay
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RBD and spike ELISAs were modified from Amanat et al. and conducted as previously described [6 (link),8 (link)]. ELISA plates were coated either with RBD (Sino Biological, Wayne, PA, USA), nucleocapsid (Gift from B. Geiss Lab) or spike protein (Sino Biological, Wayne, PA, USA). Samples and controls were added after 1 h of blocking (1X PBS, 3% milk powder, 0.1% tween). Positive controls included convalescent patient serum (gift of R. Goodrich) and monoclonal antibody CR3022 (Absolute Antibody, Boston, MA, USA). Negative control serum was charcoal inactivated pooled human serum collected in 2015 (Jackson Immuno Research, West Grove, PA, USA). Secondary antibody was added followed by development and reading via spectrophotometer (Thermo Fisher, Dallas, TX, USA).
9
SARS-CoV-2 Mimics Challenge in Mice
10
SARS-CoV-2 Spike-ACE2 Interaction Assay
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Ni-magnetic beads pull down of His-tagged S protein in complex with MBP-ACE2NTD. Ni-magnetic beads (100 μl) were washed three times with Ni binding buffer (50 mM NaH2PO4, 0.3 M NaCl, 20 mM imidazole). His-tagged Spike protein (2 μg, purchased from Sino Biological) was added to the beads pre-suspended in 200 μl of Ni binding buffer and incubated at 4°C in a roller for 30 min. Refolded MBP-ACE2NTD protein (10 μg) or equal volume of storage buffer was added to the prebound Ni-beads/S protein and co-incubation continued at 4°C for 1 h. The Ni magnetic beads were washed three times with Ni binding buffer (1 ml each time), and the protein was eluted by addition of 50 μl of Ni elution buffer (50 mM NaH2PO4, 0.3 M NaCl, 0.25 M imidazole). Approximately 17 μl of the eluted protein was loaded onto 10–20% SDS-PAG gel for analysis.
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