Potassium hexacyanoferrate (III), methanol, sodium nitrite, and hydrochloric acid were purchased from Chempur (Poland). Phosphate-buffered saline (PBS), 4-aminobenzoic acid (4-ABA), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), Tris-buffered saline (TBS), IPTG, HIS-Select® Nickel Affinity Gel, Tris-HCl, Glycerol, 4-aminothiphenol (4-ATP), glutaraldehyde (GA), bovine serum albumin (BSA), ethanol, demineralized/ultrapure water (ddH2O), hydrochloric acid (37%), sodium nitrite and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma Aldrich (Germany). Codon optimization for E. coli, ALICator Ligation Independent Cloning, B-PER Complete Bacterial Protein Extraction Reagent, Gene Art Gene Synthesis, Slide-A-Lyzer Dialysis Cassettes, Wedge Well Gel, Super Signal West Pico Plus Chemiluminescent Substrate, and 1-Step Turbo TMB-ELISA were purchased from Thermo Fisher Scientific (USA). pFastBac1 was purchased from Invitrogen (USA). 15 cm Chromatography column, Trans-Blot Turbo RTA Transfer Kit were purchased from Biorad (USA). Protein A-agarose affinity matrix was purchased from Roche (Swiss). Instant Blue was purchased from Expedeon (United Kingdom). Secondary goat anti-rabbit HRP antibodies were purchased from Jackson ImmunoResearch (United Kingdom). 96-well ELISA plate was purchased from Greiner Microlon (Germany).
1 step turbo tmb elisa
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 1-Step Turbo TMB-ELISA is a laboratory reagent used in enzyme-linked immunosorbent assay (ELISA) procedures. It is a ready-to-use substrate solution that provides a colorimetric detection of enzyme activity. The product enables a one-step, rapid detection of target analytes in ELISA experiments.
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Lab products found in correlation
Secondary antibody solution, by Jackson ImmunoResearch (3 mentions)
Ma5 11 202, by Thermo Fisher Scientific (3 mentions)
Peroxidase conjugated affinipure goat anti rabbit abs, by Jackson ImmunoResearch (2 mentions)
Ab92976, by Abcam (2 mentions)
Nanoquant microplate reader, by Tecan (2 mentions)
Goat anti mouse igg hrp secondary antibody, by Cell Signaling Technology (2 mentions)
Affinipure goat anti mouse antibodies, by Jackson ImmunoResearch (2 mentions)
Hydrochloric acid (hcl), by Chempur (1 mentions)
Sodium nitrite, by Chempur (1 mentions)
Slide a lyzer dialysis cassette, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo rta transfer kit, by Bio-Rad (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Tris hcl, by Merck Group (1 mentions)
Pfastbac1, by Thermo Fisher Scientific (1 mentions)
Potassium hexacyanoferrate 3, by Chempur (1 mentions)
B per complete bacterial protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Wedgewell gel, by Thermo Fisher Scientific (1 mentions)
His select nickel affinity gel, by Merck Group (1 mentions)
Instantblue, by Abcam (1 mentions)
Tris buffered saline (tbs), by Merck Group (1 mentions)
15 protocols using 1 step turbo tmb elisa
1
Recombinant Protein Expression and Purification
2
SARS-CoV-2 N Protein ELISA Titration
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The titration of anti-N20 antibodies was conducted similarly to the one reported in28 (link). A 96-well ELISA plate (Greiner Microlon High-Binding, clear) was coated with 100 ng/well of recombinant RNA-binding domain of N protein of SARS-CoV-2. N protein of NL63 coronavirus produced in E. coli was used as a negative control. The coated plate was incubated overnight at 4 °C. Then the plate was washed 4 × 5 min with 200 µl/well of washing buffer (Phosphate buffered saline (PBS)/0.05%Tween20), blocked for 1 h at 37 °C with 250 µl/well of blocking buffer (3% Bovine Serum Albumin (BSA)/PBS/0.05%Tween20) and washed again as described above. Then 100 µl/well of serial dilutions of rabbit anti-N20 Abs were added and incubated for 1 h at 37 °C. The plate was washed again as previously. Then 100 µl/well of a Peroxidase-conjugated AffiniPure Goat Anti-Rabbit Abs (Jackson Immuno Research) (1:2000 in 3% BSA/PBS/0.05%Tween20) was added and incubated for 1 h at room temperature. Finally, following the last plate-washing step (6 × 5 min with 200 µl/well) 100 µl/well of Horseradish Peroxidase (HRP)-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific). The plate was incubated in the dark until the blue color developed and stopped by adding 50 µL of 0.5 M sulfuric acid to each well. Absorption was measured at wavelength of 450 nm using a plate reader (TECAN).
3
Titration of Recombinant N Protein
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The titration of recombinant nucleoprotein N20 was conducted similarly to the one reported in28 (link). A 96-well ELISA plate (Greiner Microlon High-Binding, clear) was coated with serial dilutions of recombinant N protein produced in a bacterial expression system. The coated plate was incubated overnight at 4 °C. Then the plate was washed 4 × 5 min with 200 µl/well of washing buffer (PBS/0.05%Tween20) and blocked for 1 h at 37 °C with 250 µl/well of blocking buffer (3%BSA/PBS/0.05%Tween20), and the plate was washed as previously. Then 100 µl/well of rabbit anti-N20 Abs was added, incubated for 1 h at 37 °C and the plate was washed as previously. Then 100 µl/well of Peroxidase-conjugated AffiniPure Goat Anti-Rabbit Abs (Jackson Immuno Research) (1:2000 in 3% BSA/PBS/0.05%Tween20) was added, incubated for 1 h at room temperature. Finally, following the last plate-washing step (6 × 5 min with 200 µl/well) 100 µl/well of HRP-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific). The plate was incubated in the dark until the blue color developed and stopped by adding 50 µL of 0.5 M sulfuric acid to each well. Absorption was measured at wavelength of 450 nm using a plate reader (TECAN).
4
NoV-VP1 ELISA protocol for antibody detection
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Sera from the immunized mice were collected and pooled on day 56 after immunization. A 96-well ELISA plate (Greiner Microlon High-Binding, clear) was coated with 100 µl/well of L. tarentolae-derived NoV- VP1 cell lysates. The coated plate was incubated overnight at 4 °C. Next, the plates were washed 4 × 5 min with 200 µl/well of washing buffer (PBS/0.05%Tween20) and blocked for 2 h with 250 µl/well of blocking buffer (3%BSA/PBS/0.05%Tween20) at 37 °C. The plates were washed as previously and serial dilutions of mouse sera (in 3%BSA/PBS/0.05%Tween20) were added to the wells and incubated for 1 h at room temperature. Serial dilutions of rabbit anti-NoV antibodies (Abcam ab92976) ( in 3%BSA/PBS/0.05%Tween20) served as a positive control. After incubation plates were washed as previously, and appropriate secondary antibody solution (Jackson Immuno Research) (in 3%BSA/PBS/0.05%Tween20) was used for detection. Finally, following the last plate-washing step (6 × 5 min with 200 µl/well), 100 µl/well of HRP-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific), the plate was incubated in dark until the blue color developed, and the reaction was stopped by adding 50 µl of 0.5 M sulfuric acid to each well. Signal intensity at 450 nm was measured using a plate reader (NanoQuant Microplate Reader, TECAN).
5
Antibody Response to Mucins and Peptides
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Mouse sera were collected on day 56 and pooled. Antibody response was measured by ELISA assay using (i) 10 µg/ml 100 µl/well of mucins from pig gastric mucous (Porcine gastric mucin, PGM, Sigma Aldrich) in PBS (ii) 10 µg/ml 100 µl/well of biotinylated PDTRPAPGSTAPPAHGVTSA MUC1 peptide (synthesized by JPT Peptide Technologies), (iii) 100 µl/well of L. tarentolae cell lysates—WT, (iv) 100 µl/well of L. tarentolae cell lysates—NoV VP1 and (v) 100 µl/well of WT L. tarentolae cell lysates—NoV VP1-MUC1. The coated plate was incubated for 2 h with shaking at room temperature. Then the plate was washed 4 × 5 min with 200 µl/well of wash buffer (Tris buffered saline pH 7,2/0,1%BSA/0,05%Tween20). Next, 100 µl/well of pooled serum 1:100 was added, and the plate was incubated for 1 h at room temperature, then washed as previously. Then 100 µl/well of Peroxidase-conjugated anti-mouse antibody (Jackson Immuno Research; 1:2000 in 3%BSA/PBS/0,05%Tween20) was added and incubated for 1 h at room temperature. The plate was washed as previously and 100 µl/well of HRP-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific). The plate was incubated in dark until the blue color developed and the reaction was stopped by adding 50 µL of 0,5 M sulfuric acid to each well. Signal intensity was measured at 450 nm using a plate reader (Epoch, Biotek).
6
Quantification of Antibody Binding to HIV Antigens
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Micro-vinyl plates (96-well, Corning Costar, Corning, NY, USA) were coated with 0.5 µg/mL of BaL gp120 Env recombinant protein (NIH AIDS reagent #4961, Germantown, MD, USA) or Gag IIIB recombinant protein (NIH AIDS reagent #3276, Germantown, MD, USA) in sodium bicarbonate buffer and incubated overnight at 4 °C. Plates were washed three times with 0.05% PBST and blocked with 5% BSA in PBS. Mouse serum was diluted at 1:500 in PBS and incubated overnight, then washed 5 times with 0.05% PBST before goat anti-mouse IgG HRP secondary antibody (Cell signaling #7076S, Danvers, MA, USA), diluted 1:20,000 in 5% BSA in PBS, was added to each well for 2 h. After washing 6 times with 0.05% PBST, the 1-Step™ Turbo TMB-ELISA (Thermo Scientific #34022, Rockford, IL, USA) reagent was added and incubated for 20 min at room temperature. Sulfuric acid (2N) was used to stop the reaction. Plates were analyzed on a spectrophotometer and optical density was measured at 450 nm.
7
MUC1 Peptide ELISA Assay
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A 96-well ELISA plate (Pierce Streptavidin High Binding Capacity, Clear) was coated with 100 µl/well of biotinylated PDTRPAPGSTAPPAHGVTSA MUC1 peptide (synthesized by JPT Peptide Technologies) adjusted to 10 µg/ml. The coated plate was incubated for 2 h with shaking at room temperature. Then the plate was washed 4 × 5 min with 200 µl/well of wash buffer (Tris buffered saline pH 7,2/0,1%BSA/0,05%Tween20). Next, 100 µl/well of L. tarentolae cell lysates (WT, NoV VP1 and NoV VP1-MUC1 lysates) were used to coat the plate. The plate was incubated for 2 h with shaking in room temperature. The plate was washed as previously. Next, 100 µl/well of Armenian hamster anti-MUC1 antibody (MA5-11,202, Thermo Fisher Scientific; 1:100 in wash buffer) was added and the plate was incubated for 1 h at room temperature, then washed as previously. Then 100 µl/well of Peroxidase-conjugated mouse anti-Armenian hamster antibody (Santa Cruz Biotechnology; 1:2000 in wash buffer) was added and incubated for 1 h at room temperature. The plate was washed as previously and 100 µl/well of HRP-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific). The plate was incubated in dark until the blue color developed and the reaction was stopped by adding 50 µL of 0,5 M sulfuric acid to each well. Signal intensity was measured at 450 nm using a plate reader (Epoch, Biotek).
8
ELISA for MUC1 antibody detection
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Sera from immunized mice were collected and pooled on day 56 after immunization. A 96-well ELISA plate (Greiner Microlon High-Binding, clear) was coated with 100 µl/well of L. tarentolae cell lysates (WT and NoV VP1-MUC1 lysates). The coated plate was incubated overnight at 4 °C. Next, the plate was washed 4 × 5 min with 200 µl/well of washing buffer (PBS/0,05%Tween20) and blocked for 2 h with 250 µl/well of blocking buffer (3%BSA/PBS/0,05%Tween20) at 37 °C. The plate was washed as previously and serial dilutions of pooled mouse sera (in 3%BSA/PBS/0,05%Tween20) were added to the wells and incubated for 1 h at room temperature. Serial dilutions of Armenian hamster anti-MUC1 antibody (MA5-11,202, Thermo Fisher Scientific; in 3%BSA/PBS/0,05%Tween20) served as a positive control. After incubation the plate was washed as previously, and appropriate secondary antibody solution (Jackson Immuno Research; in 3%BSA/PBS/0,05%Tween20) was used for detection. Finally, following the last plate-washing step (6 × 5 min with 200 µl/well), 100 µl/well of HRP-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific), the plate was incubated in dark until the blue color developed, and the reaction was stopped by adding 50 µL of 0,5 M sulfuric acid to each well. Signal intensity at 450 nm was measured using a plate reader (Epoch, Biotek).
9
Measuring Antibody Avidity to HIV Env
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Micro-vinyl plates (96-well, Corning Costar, Corning, NY, USA) were coated with 0.5 µg/mL of BaL gp120 Env recombinant protein (NIH AIDS reagent #4961, Germantown, MD, USA) or Gag IIIB recombinant protein (NIH AIDS reagent #3276, Germantown, MD, USA) in sodium bicarbonate buffer and incubated overnight at 4 °C. Plates were washed two times with 0.05% PBST and blocked with 5% BSA in PBS. Duplicate wells containing mouse serum diluted at 1:500 in PBS were incubated overnight. The following day, diluted serum was removed and one of the duplicate wells was treated with 7M urea for 20 min, while the other well was treated with 0.05% PBST. After incubation, wells were washed 4 more times with 0.05% PBST, then incubated with goat anti-mouse IgG HRP secondary antibody (Cell signaling #7076S, Danvers, MA, USA), diluted 1:20,000 in 5% BSA in PBS for 2 h. After washing 6 times with 0.05% PBST, the 1-Step™ Turbo TMB-ELISA (Thermo Scientific #34022, Rockford, IL, USA) reagent was added and incubated for 20 min at room temperature. Sulfuric acid (2N) was used to stop the reaction. Plates were analyzed on a spectrophotometer and optical density was measured at 450 nm. The avidity index was calculated by taking the OD450 value of the urea treated well divided by the untreated well, as previously described [40 (link),41 (link)].
10
ELISA for L. tarentolae-derived VLPs
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A 96-well ELISA plate (Greiner Microlon High-Binding, clear) was coated with 100 µl/well of sequential dilutions purified L. tarentolae-derived VLPs (NoV VP1 and NoV VP1-MUC1). WT L. tarentolae cell lysate served as a negative control. The coated plate was incubated overnight at 4 °C. Next, the plate was washed 4 × 5 min with 200 µl/well of washing buffer (PBS/0,05%Tween20) and blocked for 2 h with 250 µl/well of blocking buffer (3%BSA/PBS/0,05%Tween20) at 37 °C. The plate was washed as previously and 100 µl/well of primary rabbit anti-NoV antibodies (Abcam ab92976; in 3%BSA/PBS/0,05%Tween20) and Armenian hamster anti-MUC1 antibody (MA5-11,202, Thermo Fisher Scientific) were added. After incubation the plate was washed as previously, and the appropriate secondary antibody solution (Jackson Immuno Research; in 3%BSA/PBS/0,05%Tween20) was used for detection. Finally, following the last plate-washing step (6 × 5 min with 200 µl/well), 100 µl/well of HRP-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific), the plate was incubated in dark until the blue color developed, and the reaction was stopped by adding 50 µL of 0,5 M sulfuric acid to each well. Signal intensity at 450 nm was measured using a plate reader (Epoch, Biotek).
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