Si NNs were prepared by loading 1 μg of Bev and then immersed in 1 ml of the simulated tear at 37°C. For comparison, a control group of Si NNs with Bev was stored at 4°C for 3 days. At 12 and 120 hours of release, the simulated tear was extracted for bioactivity measurement. Anti-Bev antibody HCA182 (Bio-Rad) was used as a capture antibody along with HCA184 (Bio-Rad) as a detection reagent. HCA182 (100 μl) at 1 μg ml−1 in PBS was coated overnight at 4°C on 98-well MaxiSorp black plates (Thermo Fisher Scientific) and treated with 300 μl of 5% cow serum albumin (Thermo Fisher Scientific) in PBS. Tween 20 (0.05%) (PBST) was added as a blocking agent. Both the freshly prepared Bev from 0.1 to 1000 ng ml−1 in 10% human serum (Sigma-Aldrich) in PBST and the released Bev from Si NNs were added to the well. HCA184P (100 μl) at 4 μg ml−1 in HISPEC buffer (Bio-Rad) was loaded to each well, followed by adding 100 μl of QuantaBlu (Thermo Fisher Scientific). Fluorescent signals were measured with a plate reader (Synergy Neo plate reader, BioTek Instruments) at the excitation and emission at 325 and 420 nm, respectively. The measured data were expressed as averages ± SD.
Quantablu
Manufactured by Thermo Fisher Scientific
QuantaBlu is a fluorometric assay system designed for rapid and accurate quantification of protein samples. The system utilizes a proprietary fluorescent dye that binds to proteins, enabling the measurement of protein concentrations in a simple and efficient manner. QuantaBlu provides a reliable and reproducible method for determining protein levels in a variety of sample types.
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Lab products found in correlation
5 protocols using quantablu
1
Measuring Bevacizumab Release from Silica Nanoparticles
2
Quantitative Evaluation of Angiopoietin-2 Binding
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Example 4
Binding of muteins was tested by a sandwich ELISA assay. In detail, a 384-well plate suitable for fluorescence measurements (Greiner FLUOTRAC™ 600, black flat bottom, high-binding) was coated with 20 μl of Ang-2 at a concentration of 5 μg/ml in PBS over night at 4 C. After washing, the Ang-2-coated wells were blocked with 100 μl blocking buffer containing 0.1% Tween 20 and 2% BSA (PBS-T/BSA) for 1 h at room temperature.
20 μl of serially diluted muteins were incubated in PBS-T/BSA for 1 h at room temperature (RT).
The residual supernatants were discarded and 20 μl HRP-labeled anti-Streptag antibody was added at a predetermined optimal concentration in PBS-T/BSA and incubated for 1 h at RT. After washing, 20 μl fluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well, and the reaction was allowed to proceed for 15 to 60 minutes. The fluorescence intensity of every well on the plate was read using a fluorescence microplate reader (Tecan or Molecular Devices). Curve fitting was performed using GraphPad Prism 4 software. The resulting EC50 values are summarized in Table 1 below.
3
Simultaneous Binding of Fusion Polypeptides
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Example 4
In order to demonstrate the simultaneous binding of the fusion polypeptides to HER2 and CD137, a dual-binding ELISA format was used. Recombinant HER2 (Sino Biological) in PBS (5 μg/mL) was coated overnight on microtiter plates at 4° C. The plate was washed five times after each incubation step with 80 μL PBS supplemented with 0.05% (v/v) Tween 20 (PBS-T) using a Biotek ELx405 select CW washer. The plates were blocked with 2% BSA (w/v) in PBS for 1 h at room temperature and subsequently washed again. Different concentrations of the fusion polypeptides were added to the wells and incubated for 1 h at room temperature, followed by a wash step. Subsequently, biotinylated human CD137-Fc was added at a constant concentration of 1 μg/mL in PBS-T for 1 h. After washing, Extravidin-HRP (Sigma-Adrich, 1:5000 in PBS-T) was added to the wells for 1 h. After an additional wash step, fluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well and the fluorescence intensity was detected using a fluorescence microplate reader.
The respective experimental data was plotted in
4
ELISA Assay for Protein Detection
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Protein sample (0.1 μg) in 100 μl of DPBS was coated on a 96-well plate (Corning Costar, 2592) at 4°C overnight. The plate was blocked with 200 μl of 3% non-fat milk solution in DPBST (DPBS, 0.25% Tween 20) for 2 hours at 37°C. Samples (first antibodies or phages) were incubated with 3% non-fat milk in DPBST for 2 hours at 37°C. Wells were washed four times with 200 μl of DPBST. Subsequently, horseradish peroxidase (HRP)–conjugated secondary antibody was added at a dilution in blocking solution and incubated for 1 hour at 37°C. Wells were then washed five times with 200 μl of DPBST. A 100-μl working solution of QuantaBlu (Thermo Fisher Scientific, 15169) or trimethylboron (TMB; Invitrogen, 002023) was added to each well and incubated for 10 to 30 min at room temperature before plates were read (42 (link)).
5
Quantifying Anti-3BNC117 Antibody Levels
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High binding microplates (Greiner Bio-One) were coated with 2 μg/ml of an anti-idiotipic antibody against 3BNC117 in PBS overnight at 4°C. Plates were washed with PBST, blocked for an hour with 5% BSA in PBST and washed again. For 3BNC117 IgG quantification, samples were diluted 1:50-500 fold and a standard was made using purified 3BNC117 serially diluted in PBS. For 3BNC117 isotype detection, samples were serially diluted as described in the figures. Samples and standards were incubated for an hour. Plates were then applied with HRP conjugated detection antibodies: anti-mouse IgA (Abcam), anti-mouse IgG, anti-mouse IgG1, anti-mouse IgM (Jackson ImmunoResearch) or anti mouse IgG2c (Bio-Rad Laboratories) at 2 μg/ml in PBST and were incubated for another hour. A list of antibodies used in these experiments may be found in Supplementary Table 1. Before detection with QuantaBlu (ThermoFisher) according to manufacturer protocol, plates were washed for an additional round. Detection was done in a Synergy M1 Plate reader (Biotek). When absolute quantitation is presented, the concentration of 3BNC117 was determined by reference to the dilution factor of the standard curve.
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