20μl of ectodomains (stabilized prefusion trimer) of SARS-CoV-2 or SARS-CoV, or the disulphide stabilized SARS-CoV-2 or SARS-CoV, were coated on 384 well ELISA plates at 1 ng/μl for 16 hours at 4°C. Plates were washed with a 405 TS Microplate Washer (BioTek Instruments) then blocked with 80 μl SuperBlock (PBS) Blocking Buffer (Thermo Scientific) for 1 hour at 37°C. Plates were then washed and 30 μl antibodies were added to the plates at concentrations between 4 × 10−8 and 10 ng/μl and incubated for 1 h at 37°C. Plates were washed and then incubated with 30 μl of 1/5000 diluted goat anti-human Fc IgG-HRP (invitrogen A18817). Plates were washed and then 30 μl Substrate TMB microwell peroxidase (Seracare 5120–0083) was added for 5 min at room temperature. The colorimetric reaction was stopped by addition of 30 μl of 1 N HCl. A450 was read on a Varioskan Lux plate reader (Thermo Scientific).
405 ts microplate washer
Manufactured by Agilent Technologies
Sourced in United States
The 405 TS Microplate Washer is a laboratory equipment designed for automated washing of microplates. It is capable of aspirating and dispensing liquids into microplate wells to remove unbound materials. The device operates using a microprocessor-controlled system to perform these functions.
Automatically generated - may contain errors
Lab products found in correlation
Tmb microwell peroxidase, by LGC (3 mentions)
Maxisorp plate, by Thermo Fisher Scientific (3 mentions)
Varioskan lux plate reader, by Thermo Fisher Scientific (2 mentions)
Blocker casein, by Thermo Fisher Scientific (2 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (2 mentions)
Haltr protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Superblock pbs blocking buffer, by Thermo Fisher Scientific (1 mentions)
Goat anti human fc igg hrp, by Thermo Fisher Scientific (1 mentions)
Blocker casein in tbs, by Thermo Fisher Scientific (1 mentions)
Menadione, by Merck Group (1 mentions)
Homebrew sample diluent, by Quanterix (1 mentions)
Biotek plate reader, by Agilent Technologies (1 mentions)
Hace 2 fc, by Thermo Fisher Scientific (1 mentions)
Synergy h1 multi mode reader, by Agilent Technologies (1 mentions)
Cdd h82w6, by ACROBiosystems (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Tmb substrate, by BioLegend (1 mentions)
1 n sulfuric acid, by Thermo Fisher Scientific (1 mentions)
Streptavidin coated plates, by R&D Systems (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
10 protocols using 405 ts microplate washer
1
SARS-CoV-2 Spike Protein ELISA Assay
2
SARS-CoV-2 Spike Protein ELISA
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384-well Maxisorp plates (Thermo Fisher) were coated overnight at room temperature with 3 μg/mL in 20mM Tris pH 8 and 150mM NaCl of SARS-CoV-2 S2P (Pallenson et al 2017) or SARS-CoV-2 A222V-D614G S2P, produced as previously described in Walls et al. (2020) (link). Briefly, Expi293F cells were transiently transcribed with a plasmid containing the spike protein and supernatant was clarified six days later prior to Ni Sepharose resin purification and flash freezing. Plates were slapped dry and blocked with Blocker Casein in TBS (Thermo Fisher) for one hour at 37°C. Plates were slapped dry and S2E12 (Tortorici et al., 2020 ) or S309 (Pinto et al., 2020 (link)) antibodies were serially diluted 1:3 with a starting concentration of 1000nM in TBST or NIBSC human plasma (20/130 https://www.nibsc.org/documents/ifu/20-130.pdf ) was serially diluted 1:3 starting at 1:4 of original concentration in TBST and added to the plate for one hour at 37°C. Plates were washed 4x with TBST using a 405 TS Microplate Washer (BioTek) followed by addition of 1:5,000 goat anti-human Fc IgGHRP (Thermo Fisher) for one hour at 37°C. Plates were washed 4x and TMB Microwell Peroxidase (Seracare) was added. The reaction was quenched after 1–2 minutes with 1 N HCl and the A450 of each well was read using a Varioskan Lux plate reader (Thermo Fisher).
3
Biofilm Formation and Metabolic Activity
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We used a slightly modified protocol of biofilm formation and XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] analysis, as described previously (48 (link), 49 (link)). Briefly, 5-ml cultures were grown overnight in YNB with 2% glucose at 30°C, pelleted, washed in 1× PBS, and resuspended in 1× PBS. Cells were counted on a hemocytometer, diluted to 107 cells/ml in the appropriate medium, and plated in 100-μl volumes in 96-μl polystyrene plates (avoiding edge wells). Sterile medium was plated as a negative control. Plates were incubated for 48 h at 37°C to allow for adherence and biofilm maturation. Plates were then washed three times with 200 μl of 1× PBS–0.05% Tween 20 using a BioTek 405 TS microplate washer set to an intermediate flow rate. To determine the relative levels of cells that remained after washing, we used an XTT reduction assay to quantitate metabolic activity as a proxy for viable cell density. After plate washing, 100 μl of a solution containing 0.5g/liter XTT (catalog no. X6493; Fisher) and 4 μM menadione (catalog no. 58-27-5; Sigma) in acetone in 1× PBS was added to each well. menadione was added to fresh XTT solution immediately prior to addition of the solution to a plate. Plates were incubated for 5 h before aliquots of 80 μl of supernatant were moved to a new plate for absorbance reading at 490 nm.
4
Multiplexed Immunoassay with Amplification
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Immunoassays were performed in 96-well
plates (Greiner Bio-One, 655096). For each sample to be measured,
1 × 106 beads diluted in 10 μL of Homebrew Sample
Diluent (Quanterix) were added to 100 μL of sample diluted in
Homebrew Sample Diluent. The plate was sealed and shaken at room temperature
for 1 h before washing three times with System Wash Buffer 1 (Quanterix)
using a BioTek 405 TS Microplate Washer. The beads were then resuspended
in 100 μL of detector antibody-DNA conjugate (0.6 μg/mL
diluted in Homebrew Sample Diluent) and shaken at room temperature
for 15 min. After washing 10 times with System Wash Buffer 1, the
beads were transferred to a new 96-well plate and resuspended in 60
μL of RCA reaction mix consisting of 1 U/μL phi29, 0.2
U/μL AluI, 1 mM dNTP, 0.05% Tween-20, and 1× NEBuffer 4.
The plate was shaken at 37 °C for 1.5 h, and the RCA reaction
was quenched by adding 6 μL of 500 mM EDTA before nanopore measurements
of the supernatant.
plates (Greiner Bio-One, 655096). For each sample to be measured,
1 × 106 beads diluted in 10 μL of Homebrew Sample
Diluent (Quanterix) were added to 100 μL of sample diluted in
Homebrew Sample Diluent. The plate was sealed and shaken at room temperature
for 1 h before washing three times with System Wash Buffer 1 (Quanterix)
using a BioTek 405 TS Microplate Washer. The beads were then resuspended
in 100 μL of detector antibody-DNA conjugate (0.6 μg/mL
diluted in Homebrew Sample Diluent) and shaken at room temperature
for 15 min. After washing 10 times with System Wash Buffer 1, the
beads were transferred to a new 96-well plate and resuspended in 60
μL of RCA reaction mix consisting of 1 U/μL phi29, 0.2
U/μL AluI, 1 mM dNTP, 0.05% Tween-20, and 1× NEBuffer 4.
The plate was shaken at 37 °C for 1.5 h, and the RCA reaction
was quenched by adding 6 μL of 500 mM EDTA before nanopore measurements
of the supernatant.
5
Quantifying SARS-CoV-2 Spike Protein Binding
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384-well Maxisorp plates (Thermo Fisher) were coated overnight at room temperature with 3 μg/mL of hACE2-Fc, CR3022 (Yuan et al., 2020 (link)), or S309 (Pinto et al., 2020 (link)) in 20mM Tris pH 8 and 150mM NaCl. Plates were slapped dry and blocked with Blocker Casein in TBS (Thermo Fisher) for one hour at 37°C. Plates were slapped dry and a 30-minute pre-incubated 1:5 serial dilution of mouse sera in TBST, with in initial dilution of 1:50 for hACE2-Fc competition or 1:10 for antibody competition, and a constant concentration of biotinylated (Avidity) SARS-CoV-2 S2P or SARS-CoV 2P at their EC50 values were added. Spike concentrations were 0.63nM, 5.98nM, and 0.22nM of SARS-CoV-2 S2P or 4.11nM, 2.89nM, and 0.19nM of SARS-CoV S2P for immobilized hACE2, CR3022, and S309, respectively. Plates were left for one hour at 37°C, then washed 4x with TBST using a 405 TS Microplate Washer (BioTek) followed by addition of 1:500 streptavidin-HRP (Thermo Fisher) for one hour at 37°C. Plates were washed 4x and TMB Microwell Peroxidase (Seracare) was added. The reaction was quenched after 1-2 minutes with 1 N HCl and the A450 of each well was read using a BioTek plate reader (BioTek). Data plotted and fit in Prism (GraphPad) using nonlinear regression sigmoidal, 4PL, X is log(concentration) to determine EC50 values from curve fits.
6
Rapid SARS-CoV-2 Antibody Quantification
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384-well Maxisorp plates (Thermo Fisher) were coated overnight at room temperature with 3 μg/mL of SARS-CoV-2 S2P (Pallesen et al., 2017 (link)) in 20mM Tris pH 8 and 150mM NaCl. Plates were slapped dry and blocked with Blocker Casein in TBS (Thermo Fisher) for one hour at 37°C. Plates were slapped dry and NHP sera was serially diluted 1:4 in TBST with an initial dilution of 1:4 for hACE2 competition or 1:2 for antibody competition. Random primary amine biotinylated (Thermo Fisher) hACE2-Fc, CR3022 (Yuan et al., 2020 (link)), or S309 (Pinto et al., 2020 (link)) were added, bringing the concentration of each well to the EC50 values of 0.2nM, 2nm, and 0.01nM, respectively. Plates were left for one hour at 37°C, then washed 4x with TBST using a 405 TS Microplate Washer (BioTek) followed by addition of 1:500 streptavidin-HRP (Thermo Fisher) for one hour at 37°C. Plates were washed 4x and TMB Microwell Peroxidase (Seracare) was added. The reaction was quenched after 1-2 minutes with 1 N HCl and the A450 of each well was read using a BioTek plate reader (BioTek). Data plotted and fit in Prism (GraphPad) using nonlinear regression sigmoidal, 4PL, X is log(concentration) to determine EC50 values from curve fits with upper and lower constraints determined by uncompeted ELISA per antigen.
7
KRAS Peptide Binding Assay
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The streptavidin-coated plates (R&D Systems, Minneapolis, MN) were coated with 50 μl of 0.5 μg/ml of biotinylated KRASG12V[7–16]/HLA-A3 monomer, 0.5 μg/ml biotinylated KRASWT[7–16]/HLA-A3 monomer, or 0.4 μg/ml biotinylated recombinant human CD3ε/CD3δ heterodimer (CDD-H82W6, Acro Biosystems, Newark, DE) in BAE buffer (PBS containing 0.5% bovine serum albumin [Sigma], 2 mM EDTA [Thermo] and 0.1% sodium azide) for 1 h at room temperature. Plates were washed 6 times with 1× TBST (J77500-K8, Thermo Fisher Scientific) using a 405 TS microplate washer (BioTek, Winooski, VT). scDb samples were diluted to 200 ng/ml in PBS and incubated in the coated plates for 1 h at room temperature. Plates were washed 6 times with TBST. 50 μl of 0.5 μg/ml recombinant Protein L (Pierce) was added to the well and incubated for 1 h at room temperature. Plates were washed 6 times with TBST. Plates were incubated with 50 μl of 0.2 μg/ml chicken anti-Protein L HRP antibody (ab63506, Abcam) for 1 h at room temperature and then washed 6 times with TBST. 50 μl of TMB substrate (Biolegend) was added to each well and quenched with 50 μl 1 N sulfuric acid (Fisher Scientific). Absorbance was measured at 450 and 540 nm using a Synergy H1 Multi-Mode Reader (BioTek). Reported A450 values include an A540 correction (subtract A540 from A450 for each well). All samples were measured in triplicate.
8
Peptide Array Antibody Binding Assay
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Peptide arrays were rehydrated by soaking in distilled water for 20 min. Rehydrated arrays were sprayed three times briefly with 90% isopropanol (Thermo Fisher Scientific, USA) ensuring full coverage, centrifuged to remove excess liquid, and then loaded into a peptide array cassette. Each sample was diluted 1:625 in mannitol buffer (1% mannitol in PBSTP), and then 90 μl of the diluted sample was transferred to the cassette. To facilitate antibody-peptide binding, the mixture was incubated on the arrays for 1 h at 37°C while shaking on a TeleShake95 shaker (INHECO, Martinsried, Germany). Subsequently, the cassette was washed 10 times in PBSTP with a BioTek 405TS microplate washer (BioTek Instruments, Winooski, VT, USA). Bound antibody was detected by adding 4 nM goat anti-human IgG (H+L) secondary antibody coupled to AlexaFluor 555 (Thermo Fisher Scientific, USA) in secondary incubation buffer [0.75% casein (Sigma-Aldrich, USA) in PBSTP] for 1 h at 37°C shaking on a TeleShake95 shaker. After incubation with the secondary antibody, the peptide arrays were again washed with PBSTP, followed by a rinse in distilled water. After being removed from the cassette, the peptide arrays were sprayed with 90% isopropanol and excess liquid was removed by centrifugation at 3000 RPM for 60 s.
9
Quantification of Tau Phosphorylation Biomarkers
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Cerebrospinal uid levels of p-tau (Ser 181) and p-tau (Thr 205) were determined using a Singulex Erenna immunoassay platform (USA) (Hastings et al. 2017 ). HT7 was adopted as capture antibody. AT270 and AT8 which separately recognized p-tau (Ser 181) and p-tau (Thr 205) served as detection antibodies. In the process, the measurement was performed in the presence of HaltR Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher). Brie y, in 96-well plate, each well contained 10 mg capture beads, 50 μl Singulex assay buffer and 50 μl cerebrospinal uids (or standard peptide), which was diluted in PBS-T (0.2% Tween 20 in PBS). The plate was incubated at 25℃ for 4 hours, and was washed using a Biotek 405 TM TS microplate washer. Then, 20 μl AT270 or AT8 antibody (50 ng/ml) was added into the plate, and was incubated and shaken overnight at 25℃. The cerebrospinal uid specimen was developed according to the instruction, and the result was analyzed using Sgx link software (Singulex Erenna).
10
Quantification of Tau Phosphorylation Biomarkers
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Cerebrospinal uid levels of p-tau (Ser 181) and p-tau (Thr 205) were determined using a Singulex Erenna immunoassay platform (USA) (Hastings et al. 2017 ). HT7 was adopted as capture antibody. AT270 and AT8 which separately recognized p-tau (Ser 181) and p-tau (Thr 205) served as detection antibodies. In the process, the measurement was performed in the presence of HaltR Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher). Brie y, in 96-well plate, each well contained 10 mg capture beads, 50 μl Singulex assay buffer and 50 μl cerebrospinal uids (or standard peptide), which was diluted in PBS-T (0.2% Tween 20 in PBS). The plate was incubated at 25℃ for 4 hours, and was washed using a Biotek 405 TM TS microplate washer. Then, 20 μl AT270 or AT8 antibody (50 ng/ml) was added into the plate, and was incubated and shaken overnight at 25℃. The cerebrospinal uid specimen was developed according to the instruction, and the result was analyzed using Sgx link software (Singulex Erenna).
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