Ninety‐six well polystyrene plates NUNC MaxiSorp™ (Thermo Fisher Scientific) were coated for 1 hr at 37°C with recombinant human ACE2, with human recombinant nerve growth factor β (R&D Systems) and with bovine histone III (Sigma‐Aldrich). All proteins were diluted to 10 μg/ml in PBS. After, the plates were blocked with 0.25% solution of Tween 20 in PBS. S proteins from Wuhan and Omicron variants were diluted to final concentration of 1 μg/ml in T‐PBS or in T‐PBS containing fetal calf serum at two‐fold dilutions in the range from 1/2 to 1/1052‐fold diluted (50 to 0.097%). The S proteins were pre‐incubated for 10 min at room temperature and then transferred to plates with immobilized ACE2 and incubated for 1 hr at 37°C. Next steps of ELISA assay were identical to the ones described with sole exception that instead of o‐phenylenediamine dihydrochloride as a peroxidase substrate in these experiments was used TMB high sensitivity substrate solution (BioLegend, San Diego, CA) and absorbance was recorded at 450 nm.
Tmb high sensitivity substrate solution
Manufactured by BioLegend
Sourced in United States
TMB High Sensitivity Substrate Solution is a laboratory reagent used for the detection and quantification of horseradish peroxidase (HRP) in various immunoassay applications. This solution provides a sensitive colorimetric reaction that can be measured spectrophotometrically.
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Lab products found in correlation
Bovine histone 3, by Merck Group (1 mentions)
Nunc maxisorp, by Thermo Fisher Scientific (1 mentions)
Acidic stop solution, by Southern Biotech (1 mentions)
Wallac victor plate reader, by PerkinElmer (1 mentions)
Tmb stop solution, by BioLegend (1 mentions)
Elisa wash buffer, by BioLegend (1 mentions)
Hrp donkey anti rabbit igg, by BioLegend (1 mentions)
Maxisorp, by Thermo Fisher Scientific (1 mentions)
5 protocols using tmb high sensitivity substrate solution
1
SARS-CoV-2 S Protein Binding Assay
2
ELISA-Based Quantitation of Anti-SIV Antibodies
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Antibody responses against p27 and gp41 were quantitated by regular ELISA using SIVmac239 p27 purified recombinant protein (Catalog# 13446; obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH) and recombinant SIV gp41 (Catalog# 5019; ImmunoDX) to coat plates at 10 μg/ml in PBS. Plates were then washed, blocked and incubated for 1 h at 37°C with a 1:20 dilution of the corresponding monkey sera or 5L7 antibody diluted to 2 μg/ml. Antibodies binding the p27 antigen or the gp41 were detected with a goat anti-monkey secondary antibody (Catalog# 2015-05, Santa Cruz) and developed with TMB High Sensitivity Substrate Solution (Catalog# 421501, Biolegend). The reaction was stopped by the addition of 50 μl of acidic stop solution (SouthernBiotech, Birmingham, AL), and the optical density at 450 nm was measured using a Wallac Victor plate reader (Perkin-Elmer, Waltham, MA).
3
DPCC ELISA Assay for Top1-DPCC Detection
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Following DPCC adsorption, all washes and incubations were performed on a plate shaker at room temperature. Wells were washed three to four times with 150–200 μl 1× PBS and blocked for 1 h in 100 μl of 1× ELISA Assay Diluent. Samples were incubated with 60 μl primary antibodies for 2–3 h; washed four times with 150 μl 1× ELISA Wash Buffer (Biolegend) or PBST (PBS containing 0.1% Tween-20); incubated with 60 μl secondary HRP-conjugated antibodies for 30–45 min and washed four times with 150 μl 1× ELISA Wash Buffer or PBST. Following addition of 100 μl of TMB High Sensitivity Substrate Solution (Biolegend), plates were incubated in the dark for 20 min and the reaction was stopped by addition of 50 μl TMB Stop Solution (Biolegend). Absorbance was read at 450 nm, and the A450 reading was corrected for background by subtraction of A570. Samples were run in duplicates or triplicates, and corrected for background by subtraction of signal from control wells that contained no DNA. The DPCC ELISA assay readily detects Top1-DPCC using samples prepared from human cells by lysis in RLT, DZ or LS1 containing 3.75 M GTC and 2 M LiCl, with no apparent difference in signal. DNA isolates contained little if any protease activity so that samples stored 6 months at 4°C did not exhibit substantial loss of immunoreactivity
4
Periplasmic Expression and ELISA of Viral Proteins
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The pMAL-p5X vector has the signal peptide on pre-MBP directs fusion proteins to the periplasm; for fusion proteins that can be successfully exported to the periplasm of E. coli. Sixteen microliter of periplasmic protein (2.4 mg/ml) or gp350/220 protein (1 mg/ml) (MyBioSourceeach) was diluted with 80 ul of coating buffer, put on 96 well plate and incubated overnight at 4 °C. The solution was discarded and washed with PBST for 5 minutes three separate times. For each well, we added 200 μL blocking buffer (0.25% gelatin in PBST 0.2%) for 1 hour at room temperature. The solution was discharged and washed with PBST for 5 minutes three separate times. Rabbit polyclonal anti-gp350 (Catalog number: 142971, NovoPro) diluted with PBST (1:5000) was added to wells, and incubated for 1 hour at room temperature. The solution was then discharged and washed with PBST for 5 minutes three separate times. HRP Donkey anti-rabbit IgG (Catalog number: 406401, BioLegend) diluted with PBST (1:80.000) was added to wells, and incubated for 1 hour at room temperature. After washing three separate times, 100 ul TMB High Sensitivity Substrate Solution (Catalog number: 421501, BioLegend) was added to wells for 2 min, followed by the addition of 50 ul of stop solution. The absorbance was read at 450 nm via ELISA Reader ELx 808 BioTek.
5
Galectin-3 Binding to Native and Modified Mucins
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Native and modified mucins were adsorbed onto a 96-well plate (Maxisorp; Thermo Fisher Scientific, Waltham, MA, USA) at 10 µg/ml in PBS overnight at 4°C. Wells were washed with PBS containing 0.05% Tween-20 and blocked with 1% bovine serum albumin in PBS for 2 h. Wells were sequentially washed and incubated with 5 µg/ml of Gal-3 (diluted in blocking buffer) for 2 h at 37°C. For the inhibition assays, Gal-3 was prepared in blocking buffer containing 100 mM lactose. After further washes, a mouse monoclonal anti-Gal-3 was added at a 1:5000 dilution in blocking buffer and incubated at 37°C for 1 h. Wells were washed as above and goat anti-mouse IgG horseradish peroxidase conjugate was added to each well at a 1:5000 dilution and incubated for 45 min at 37°C. After final washes, the presence of Gal-3 was detected by addition of TMB High Sensitivity Substrate Solution (BioLegend, San Diego, CA, USA). The colorimetric reaction was stopped by adding an equal volume of 2 N sulfuric acid (H2SO4) and read at 450 nm with a Benchmark Plus Microtiter Plate Reader (Bio-Rad, Hercules, CA, USA).
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