Microtiter plates (Costar 3690) were incubated with coating antigen BSA-KET in PBS (5 μg/mL, 25 μL) (18 h, 37 °C). 5% Non-fat milk in PBS (30 min, 37 °C) was added to block non-specific binding. Mouse sera in 5% non-fat milk were serially diluted across the plate before incubation in a moist chamber (1 h, 37 °C). The plate was washed 20 times with PBS before incubation with horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG in a moist chamber (30 min, 37 °C). The plate was further washed 20 times with PBS before being developed with the TMB substrate kit (Thermo Pierce) and the absorbance at 450 nm measured on a microplate reader (SpectraMax Devices). Titers were calculated as the dilution corresponding to 50% of the maximum absorbance from a plot of the absorbance versus log(dilution) using GraphPad Prism 9. Sera were pooled across biological samples from each group, and all studies were completed in at least technical triplicate for each time-point.
Tmb substrate kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TMB substrate kit is a laboratory reagent used for the detection and quantification of enzyme-linked immunosorbent assays (ELISA). It contains a chromogenic substrate, 3,3',5,5'-Tetramethylbenzidine (TMB), which undergoes a color change in the presence of an enzyme, enabling the visualization and measurement of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Nunc maxisorp plate, by Thermo Fisher Scientific (4 mentions)
Microtiter plate, by Corning (3 mentions)
Peroxidase conjugated donkey anti mouse igg, by Jackson ImmunoResearch (3 mentions)
Victor3 plate reader, by PerkinElmer (3 mentions)
Spectramax m2e, by Molecular Devices (2 mentions)
96 well plate, by Thermo Fisher Scientific (2 mentions)
Maxisorp immunoplate, by Thermo Fisher Scientific (2 mentions)
Sunrise absorbance reader, by Tecan (2 mentions)
Hrp conjugated goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Klh gp61, by GenScript (2 mentions)
Goat anti mouse igg h l secondary antibody conjugated to hrp, by Thermo Fisher Scientific (2 mentions)
Elisa plate, by Greiner (2 mentions)
C 3041, by Merck Group (2 mentions)
Affinipure alpaca anti human igg h l, by Jackson ImmunoResearch (2 mentions)
Microplate reader, by Agilent Technologies (2 mentions)
Human ifn α2 recombinant protein, by Miltenyi Biotec (2 mentions)
Human ifn ω recombinant protein, by Thermo Fisher Scientific (2 mentions)
Rabbit anti mouse igg cross adsorbed secondary antibody conjugated to hrp, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Np23 bsa, by Biosearch Technologies (2 mentions)
47 protocols using tmb substrate kit
1
Enzyme-linked Immunosorbent Assay for Antibody Titer Determination
2
Anti-MA IgG Production by ELISA
3
Anti-Nicotine IgG Quantification by ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Production of anti-nicotine IgG was evaluated
by ELISA. Costar 3690 microtiter plates were incubated with AM1-BSA
in PBS (5 μg/mL, 25 μL) at 37 °C and the solution
allowed to evaporate, before MeOH fixation. Nonspecific binding was
blocked with 5% nonfat milk in PBS (30 min, 37 °C). Mouse plasma
in 1% BSA (25 μL) was serially diluted across the plate before
incubation in a moist chamber (1.5 h, 37 °C). After washing with
dH2O, peroxidase-conjugated donkey antimouse IgG (Jackson
ImmunoResearch Laboratories, Inc.) was added and the plates incubated
in a moist chamber (30 min, 37 °C). After further washing with
dH2O, plates were developed with the TMB substrate kit
(Thermo Pierce) and the absorbance at 450 nm measured on a SpectraMax
M2e Molecular Devices microplate reader. Titers were calculated
as the dilution corresponding to 50% of the maximum absorbance from
a plot of the absorbance versus log(dilution) using GraphPad Prism
5.
by ELISA. Costar 3690 microtiter plates were incubated with AM1-BSA
in PBS (5 μg/mL, 25 μL) at 37 °C and the solution
allowed to evaporate, before MeOH fixation. Nonspecific binding was
blocked with 5% nonfat milk in PBS (30 min, 37 °C). Mouse plasma
in 1% BSA (25 μL) was serially diluted across the plate before
incubation in a moist chamber (1.5 h, 37 °C). After washing with
dH2O, peroxidase-conjugated donkey antimouse IgG (Jackson
ImmunoResearch Laboratories, Inc.) was added and the plates incubated
in a moist chamber (30 min, 37 °C). After further washing with
dH2O, plates were developed with the TMB substrate kit
(Thermo Pierce) and the absorbance at 450 nm measured on a SpectraMax
M2e Molecular Devices microplate reader. Titers were calculated
as the dilution corresponding to 50% of the maximum absorbance from
a plot of the absorbance versus log(dilution) using GraphPad Prism
5.
4
Antibody Titer Determination via ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Microtiter plates (Costar 3690) were
incubated with coating antigen, hapten-BSA, 5 μg/mL, 25 μL/well
at 4 °C overnight. The plates were then blocked by 5% nonfat
milk in PBS, 50 μL/well, at room temperature for 45 min to get
rid of nonspecific binding. The blocking buffer was dumped out. Mouse
sera were diluted 1:100 in 1% BSA PBS solution. When titering, 25
μL of mouse serum was serially diluted 1:1 in 1% BSA PBS buffer
(pH 7.4) across 12 columns starting at 1:200. The plates were incubated
in a moist chamber at 37 °C for 2 h followed by washing 10 times
using a shower head with dH2O before adding secondary antibodies.
Peroxidase-conjugated donkey antimouse IgG (Jackson ImmunoResearch
Laboratories, Inc.; Catalog # 715-035-151) was diluted 1:10 000
for ELISA according to the instructions. Secondary antibodies were
added at 25 μL/well, and the plates were incubated in a moist
chamber at 37 °C for 1 h. The plates were further washed 10 times
with dH2O. Each well was treated with 50 μL of developing
reagent, TMB substrate kit (Thermo Pierce), waiting for blue color
to develop over 10 min before being quenched by 50 μL of 2 M
H2SO4. Absorbance was read on a microplate reader
(SpectraMax M2e Molecular Devices) at 450 nm. In GraphPad PRISM 8,
absorbance values were fit using the log (inhibitor) vs normalized
response–variable slope equation to determine midpoint titer.
incubated with coating antigen, hapten-BSA, 5 μg/mL, 25 μL/well
at 4 °C overnight. The plates were then blocked by 5% nonfat
milk in PBS, 50 μL/well, at room temperature for 45 min to get
rid of nonspecific binding. The blocking buffer was dumped out. Mouse
sera were diluted 1:100 in 1% BSA PBS solution. When titering, 25
μL of mouse serum was serially diluted 1:1 in 1% BSA PBS buffer
(pH 7.4) across 12 columns starting at 1:200. The plates were incubated
in a moist chamber at 37 °C for 2 h followed by washing 10 times
using a shower head with dH2O before adding secondary antibodies.
Peroxidase-conjugated donkey antimouse IgG (Jackson ImmunoResearch
Laboratories, Inc.; Catalog # 715-035-151) was diluted 1:10 000
for ELISA according to the instructions. Secondary antibodies were
added at 25 μL/well, and the plates were incubated in a moist
chamber at 37 °C for 1 h. The plates were further washed 10 times
with dH2O. Each well was treated with 50 μL of developing
reagent, TMB substrate kit (Thermo Pierce), waiting for blue color
to develop over 10 min before being quenched by 50 μL of 2 M
H2SO4. Absorbance was read on a microplate reader
(SpectraMax M2e Molecular Devices) at 450 nm. In GraphPad PRISM 8,
absorbance values were fit using the log (inhibitor) vs normalized
response–variable slope equation to determine midpoint titer.
5
Antibody Titer and Competitive ELISA
6
Quantification of OVA-specific IgE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Levels of OVA-specific IgE in serum or BAL fluid were determined by ELISA. Briefly, sample wells of a Nunc maxisorb ELISA plate (Nunc, Wiesbaden, Germany) were coated with 5 μg/ml OVA overnight and then blocked with 1% bovine serum albumin. After incubation with diluted samples, bound OVA-specific antibodies were detected with biotinylated rat anti-mouse IgE (clone R35–72, BD Biosciences). The biotinylated antibody was detected by horseradish peroxidase conjugated extravidin (Sigma, St. Louis, MO) and a TMB substrate kit (eBioscience). A standard curve was generated with a commercial anti-OVA IgE standard (clone 2C6; AbD Serotec, Puchheim, Germany).
7
Serological Profiling of Anti-rOmpB-4 Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood samples were collected from the tail veins of mice per immunized group and pooled together on day 7, 14, 21, and 28 after primary immunization, respectively. Anti-rOmpB-4 IgGs were detected by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plate (Nunc, Shanghai, China) was coated with 1.5 μg/ml rOmpB-4 overnight and incubated with mouse sera at the dilution of 1:1000. Then, the IgG, IgG1, or IgG2a to rOmpB-4 was determined with goat anti-mouse IgG, IgG1, or IgG2a HRP-conjugated antibodies (1:5000) and a TMB substrate kit (eBioscience, San Diego, CA) according to previous methods [32 (link)]. Absorbance at 450nm was analysed with a UVM 340 microplate reader (Asys Hitech GmbH, Eugendorf, Austria). Anti-C. burnetii phase I/II IgGs were detected by indirect immunofluorescence assay (IFA) as per methods described previously [37 (link)]. The phase I or II C. burnetii-coated slide was incubated with sera from mice immunized with rOmpB-4 mixed with CMR at two-fold dilution (initial at the dilution of 1:100) in PBS for 45 min at 37°C. After three washes with PBS, the C. burnetii cells on the slides were incubated with a 1:100 dilution of FITC-conjugated goat anti-mouse IgGs (eBioscience, San Diego, CA) for 45 min at 37°C. After another three washes, the coxiella cells on the slides were observed under a fluorescence microscope (Olympus BX60).
8
SDS-PAGE Analysis of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS-PAGE was carried out under denaturing and non-denaturing conditions. Under denaturing conditions, the purified protein was mixed with 2× sample buffer (100 mM Tris–HCl (pH 6.8), 4% SDS, 20% glycerol, 0.2% bromphenol blue) containing 200 mM dithiothreitol (DTT) and boiled for 5 min at 95 °C. In contrast, non-denaturing SDS-PAGE was performed according to the protocol reported by Bender et al. (2005) (link). Briefly, the purified protein was mixed with 2× sample buffer containing 0.4% SDS (in the absence of a reducing agent) and loaded directly onto a gel (without boiling of the sample). The protein was separated by 5–20% polyacrylamide gradient gel (e-PAGEL, ATTO) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was incubated in 5% skim milk (Wako) in T-PBS buffer (phosphate-buffered saline, pH 7.2 (PBS) containing 0.05% Tween 20) for 60 min at 37 °C, and then incubated with a mAb against S1 protein in 5% skim milk in T-PBS buffer. Next, the membrane was incubated with a peroxidase-conjugated second antibody, goat anti-mouse IgG (H + L) (Jackson). The reacted protein was visualized using a TMB substrate kit (Invitrogen).
9
Serum Antibody Titers in Vaccinated Chickens
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood serum from all vaccine groups (N = 9/group) were tested for antibody response compared to the non-vaccinated (PBS) group collected on day 29 post-vaccination. Serum samples were diluted 1:200 in SEA blocking buffer and added to an ELISA plate coated with 2 μg/ml of individual rAg in coating buffer, serially diluted 1:2, and incubated for 1 hour at room temperature. Rabbit-anti-chicken-IgY-HRP antibody (Sigma) was used with the TMB substrate kit (Invitrogen). The relative antibody titer was determined as the reciprocal of the highest dilution that gave an absorbance at 450 nm twice of the control.
10
Quantifying PD-L1 and PD-1 Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 96-well plated were coated overnight with the indicated antigens at 2 or 5 μg/ml, washed three times with PBS-T, and blocked with 4% nonfat dried milk in PBS for 1 h at room temperature. Twofold serial dilution of biotin-labeled anti-mPD-L1 antibody (BioLegend, San Diego, CA, USA) from 6 μg/ml was measured. For mPD-1, which was biotinylated according to manufacturer’s instructions (Genemore, Shanghai, China), it was added at 25, 50, and 100 μg/ml and incubated for 1 h at room temperature. The wells were washed and avidin-conjugated HRP (1:4,000) in 0.5% BSA was added and incubated for 1 h at room temperature. A TMB substrate kit (Invitrogen, Waltham, MA, USA) was used for detection at 450 nm. IFN-γ levels in the supernatants were determined by sandwich ELSA assays (BioLegend, CA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!