ELISAs were performed according to standard protocols. Briefly, 384-well Maxisorp plates were coated with NeutrAvidin (10 μg/ mL) overnight at 4 °C and subsequently blocked with BSA (2% w/v) for 1 h at 20 °C. 10−100 nM pY antigens were captured on the NeutrAvidin-coated wells for 30 min followed by the addition of various concentrations of phage or recombinant antibodies for 30 min. The secondary Abs were either a horseradish peroxidase (HRP)-conjugated anti-M13 phage antibody (Sino Biological) for phage ELISA or an antihuman-IgG antibody (Sigma-Aldrich) for recombinant protein ELISA. The ELISA plates were washed three times after each incubation, and Ab binding was detected by TMB substrate (VWR) and read at 450 nm.
Tmb substrate
Manufactured by Avantor
Sourced in United States
TMB substrate is a colorimetric substrate used in enzyme-linked immunosorbent assay (ELISA) applications. It is a ready-to-use solution that can be directly added to ELISA plates to facilitate the detection and quantification of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Anti m13 hrp conjugate, by Sino Biological (3 mentions)
M200 pro spectrophotometer, by Tecan (3 mentions)
Nunc maxisorp flat bottom clear plates, by Thermo Fisher Scientific (3 mentions)
Anti human igg antibody, by Merck Group (1 mentions)
Goat anti human igg hrp, by Southern Biotech (1 mentions)
Versamax microplate reader, by Molecular Devices (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg, by Proteintech (1 mentions)
0.22 m filter, by Merck Group (1 mentions)
Hrp conjugated anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Prestained protein marker, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
9 protocols using tmb substrate
1
Quantifying Antibody-Antigen Interactions via ELISA
2
Measurement of AvFc Variant Binding
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Recombinant envelope glycoprotein gp120 from HIV‐1 (CM235, NIH ARP 12816) was coated overnight at 4 °C at 0.3 μg/mL in carbonate buffer, pH 9.5. After coating, wells were blocked with PBST containing 5% nonfat dry milk for 1 h at 37 °C. AvFc variants were then incubated on the plate beginning at 13 nM with 1 : 5 serial dilutions for 1 h at 37 °C, followed by detection with a 1 : 10 000 dilution of goat anti‐human IgG‐HRP (2040‐05; Southern Biotech, Birmingham, AL). Plates were developed for 5 min with TMB substrate (VWR 95059‐286), with development stopped with an equal volume of 2 N sulphuric acid and plates read at 450 nm. Dose–response curves were fit with 4‐parameter nonlinear regression in GraphPad Prism which was used to calculate EC50 values.
3
Phage Display Screening for Spike-RBD Binders
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For each phage clone, 4 different conditions were tested—Direct: Spike-RBD-Fc, Competition: Spike-RBD-Fc with an equal concentration of Spike-RBD-Fc in solution, Negative selection: ACE2-Fc/Spike-RBD-Fc complex, and Control: Fc. 384-well Nunc Maxisorp flat-bottom clear plates (Thermo Fisher Scientific) were coated with 0.5 μg/mL of NeutrAvidin in PBS overnight at 4°C and subsequently blocked with PBS + 0.02% Tween-20 + 2% BSA for 1 hr at room temperature. Plates were washed 3X with PBS containing 0.05% Tween-20 (PBST) and were washed similarly between each of the steps. 20 nM of biotinylated Spike-RBD-Fc, ACE2-Fc/Spike-RBD-Fc complex, or Fc diluted in PBSTB was captured on the NeutrAvidin-coated wells for 30 min, then blocked with PBSTB + 10 μM biotin for 30 min. Phage supernatant diluted 1:5 in PBSTB were added for 20 min. For the competition samples, the phage supernatant was diluted into PBSTB with 20 nM Spike-RBD-Fc. Bound phage were detected by incubation with anti-M13-HRP conjugate (Sino Biological)(1:5000) for 30 min, followed by the addition of TMB substrate (VWR International). The reaction was quenched with the addition of 1 M phosphoric acid and the absorbance at 450 nm was measured using a Tecan M200 Pro spectrophotometer.
4
ELISA-Based Viral Antibody Detection
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Suspensions of purified virions were coated onto 96-well ELISA plates at 4 µg total protein per mL in PBS (50 µL per well) and incubated at 4 degrees C overnight. Excess protein was decanted, and plates washed five times with 0.1% Tween-20 in PBS (wash buffer). Plates were blocked with 3% nonfat milk in wash buffer for 1 h. Mid-titer rabbit polyclonal serum obtained from BEI Resources (Manassas, VA, USA) was 2-fold serially diluted in 1% nonfat milk in wash buffer, applied to plates, and incubated for 2 h. Antisera was decanted and plates were washed as above, followed by incubation for 1 h at room temperature with a 1:3000 dilution of goat-anti-rabbit secondary antibody conjugated to HRP in wash buffer with 1% nonfat milk. The extent of antibody capture was measured by colorimetric detection following treatment with TMB substrate (VWR Scientific, Radnor, PA, USA) and acid stop solution and quantified on a VERSAMax microplate reader (Molecular Devices, San Jose, CA, USA).
5
Phage Display Screening for Spike-RBD Binders
6
Phage Screening for Spike-RBD Binders
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For each phage clone, 4 different conditions were tested—Direct: Spike-RBD-Fc, Competition: Spike-RBD-Fc with equal concentration of Spike-RBD-Fc in solution, Negative selection: ACE2-Fc/Spike-RBD-Fc complex, and Control: Fc. 384-well Nunc Maxisorp flat-bottom clear plates (Thermo Fisher Scientific) were coated with 0.5 μg/mL of NeutrAvidin in PBS overnight at 4°C and subsequently blocked with PBS + 0.02% Tween-20 + 2% BSA for 1 hr at room temperature. Plates were washed 3X with PBS containing 0.05% Tween-20 (PBST) and were washed similarly between each of the steps. 20 nM of biotinylated Spike-RBD-Fc, ACE2-Fc/Spike-RBD-Fc complex, or Fc diluted in PBSTB was captured on the NeutrAvidin-coated wells for 30 min, then blocked with PBSTB + 10 μM biotin for 30 min. Phage supernatant diluted 1:5 in PBSTB were added for 20 min. For the competition samples, the phage supernatant was diluted into PBSTB with 20 nM Spike-RBD-Fc. Bound phage were detected by incubation with anti-M13-HRP conjugate (Sino Biological)(1:5000) for 30 min, followed by addition of TMB substrate (VWR International). The reaction was quenched with addition of 1 M phosphoric acid and the absorbance at 450 nm was measured using a Tecan M200 Pro spectrophotometer.
7
Indirect ELISA for HLA-F Reactivity
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Indirect ELISA was used to determine the immune reactivity of HLA-F and to screen positive hybridoma cells as described previously 28 (link). Briefly, ELISA plates were plated with purified HLA-F protein in PBS (pH 7.4), coated at 4 °C overnight and. After which, the plates were incubated with 100 μL diluted antibodies at 37 °C for 1 h, and followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Proteintech Group, China) at a 1:5000 dilution in PBST at 37 °C for 1 h. After washing, 50 μL/well of TMB substrate (Amresco, Solon, Ohio, USA) was added into the wells, and the plates were incubated for 15 min at room temperature in the dark. OD450 values were obtained after stopping the reaction with 1 M HCl (100 μL/well).
8
Extraction and Analysis of Bacterial Proteins
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Whole cell proteins (WCPs) and extracellular proteins (ECPs) were extracted and concentrated (Yin et al. 2018 (link)). Overnight cultures of E. piscicida were sub-cultured into 50-mL fresh DMEM and statically incubated for 24 h to 72 h at 28 °C; bacteria were then harvested by centrifugation at 5,000 × g for 10 min at 4 °C for WCPs. For ECPs, culture supernatants were filtered with 0.22-µm filters (Millipore, USA), and concentrated using 10 kDa cutoff centrifugal filter devices (Millipore, USA). Proteins were separated by 12% SDS-PAGE, followed by Coomassie blue staining. For western blots, separated proteins were wet transferred onto PVDF membranes (Millipore, USA) and incubated with a 1:1000 dilution of mouse anti-EseB (GL Biochem, China). HRP-conjugated anti-mouse IgG (Santa Cruz Biotechnology, CA) was used at a 1:2,000 dilution as a secondary antibody. Proteins were visualized with TMB substrate (Amresco, USA). Mouse anti-DnaK (Santa Cruz Biotechnology, USA) was used as a loading control.
9
SBV Protein Characterization by SDS-PAGE and Western Blotting
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SDS-PAGE and western blotting was carried out to confirm SBV isolate and antigen preparation method and to determine SBV-seropositive serum samples were reacting against viral proteins. For separation of protein suspensions, protein electrophoresis was carried out in 10% SDS-PAGE gel. The proteins were transferred onto a PVDF membrane (Thermo Scientific, Waltham, Massachusetts, USA) and the membrane was blocked overnight at 4°C with 5% skimmed milk powder in phosphate buff ered saline with 0.05% Tween-20 (PBST). SBV-seropositive and -seronegative cattle sera were used as primary antibody and the membrane was incubated in sera diluted 1:100 in 0.01% PBST at room temperature for 2 h. Washing step was carried out with 0.1% PBST for three times. Rabbit anti-bovine IgG HRP secondary antibody (Life Technologies, Carlsbad, California, USA) diluted 1:1000 in 0.01% PBST was added onto the membrane and incubated at room temperature for 1 h. Following washing with 0.1% PBST, TMB substrate (Amresco, Solon, Ohio, USA) was poured onto the membrane, incubated for 5 min and evaluated with pre-stained protein marker (Thermo Scientific, Waltham, Massachusetts, USA).
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