After coating with FN, a monoclonal antibody for the FNIII12-14 domain (also known as Heparin II domain) was used (Santa Cruz Biotechnologies, sc-18827) in dilution 1:30 at 37 °C for 2 h. Samples were washed three times with PBS/0.5% Tween 20. An anti-mouse IgG horse radish peroxidase (HRP)-conjugated antibody (Invitrogen, 626520) was then used in dilution 1:2000 at room temperature for 1 h. After washing twice, samples were exposed to the substrate solution (R&D, DY999) for 20 min at room temperature in the dark. A stop solution (R&D, DY994) was added before reading the absorbance at 450 nm.
Dy999
Manufactured by R&D Systems
Sourced in United States
The DY999 is a versatile laboratory instrument designed for a range of applications. It features precise temperature control and advanced measurement capabilities. The core function of the DY999 is to provide accurate and reliable data acquisition for various experimental and analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Dy994, by R&D Systems (13 mentions)
Wa126, by R&D Systems (7 mentions)
Dy990, by R&D Systems (7 mentions)
Dy995, by R&D Systems (6 mentions)
Dy268, by R&D Systems (6 mentions)
Dy992, by R&D Systems (6 mentions)
Dy006, by R&D Systems (6 mentions)
248 n4 250 cf, by R&D Systems (4 mentions)
Dy998, by R&D Systems (4 mentions)
Streptavidin hrp, by R&D Systems (2 mentions)
Bicinchoninic acid (bca), by Abcam (2 mentions)
Superblock blocking buffer, by Thermo Fisher Scientific (2 mentions)
Stop solution, by Cell Signaling Technology (2 mentions)
Biotinylated goat anti human vegf, by R&D Systems (2 mentions)
Opsys mr system plate reader, by Dynex (2 mentions)
Filtered centrifree ultra ltration device, by Merck Group (2 mentions)
Maxisorp, by Thermo Fisher Scientific (1 mentions)
Hepatocyte growth factor (hgf), by R&D Systems (1 mentions)
Abts elisa development kit, by Thermo Fisher Scientific (1 mentions)
Elisa development kit, by Thermo Fisher Scientific (1 mentions)
28 protocols using dy999
1
Quantitative Analysis of FNIII12-14 Domain
2
Quantifying Bacterial Protein Expression
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100 µl of bacterial culture adjusted to OD600 = 1 in a carbonate coating buffer (0.1 M NaHCO3, 0.1 M Na2CO3; pH to 9.5) were inoculated into 96 well microtiter plate MaxiSorp (Nunc.) and incubated overnight at 4 °C. Bacterial culture was removed by inversion, washed with T-PBS (×3) and incubated with 2% Milk/T-PBS for 1 hour at room temperature. Primary antibodies were diluted 1/100 for anti-YeeJ and 1/1000 for anti-E. coli in 1% Milk/T-PBS and incubated 1 hour at RT and washed with T-PBS (×3). Secondary antibody anti-rabbit IgG HRP was diluted 1/10000 in 1% Milk/T-PBS, incubated 1 hour at RT and washed with T-PBS (×3). Substrate reagent R&D Systems DY999 and Stop solution R&D Systems DY994 were used to develop and absorbance was read at OD420. The results are averages for four replicate wells in three independent experiments.
3
ELISA to Study HGF-Glypican-3 Inhibition
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We designed an ELISA to study the ability of ILB® to inhibit the interaction between HGF and Glypican-3 (a cell surface heparan sulphate proteoglycan) (Gao et al., 2015 (link)). The bound Glypican-3 (biotinylated) in the system is measured. The amount of Glypican signal lost (relative to the expected binding) is a measure of the ability of ILB® to mobilize HGF from its binding to Glypican-3.
Briefly, the ELISA plate was coated with HGF (0.2 μg/ml, 100 µl/well, R&D Systems, Abingdon, United Kingdom) overnight at 4°C. Glypican-3 (R&D Systems) binding was quantified after 30 min over a range of concentrations in the absence (vehicle) or presence of a range of concentrations of ILB®. After three washes with PBS, streptavidin-HRP (according to manufacturer’s recommendations for the batch used, R&D Systems) was added to the wells for 1 h to label the bound Glypican-3. After a further 3 washes, the remaining HRP activity was quantified using an HRP substrate (DY999, R&D Systems) at 450 and 540 nm, with Glypican-3 levels determined by reference to a standard calibration curve. All conditions were assayed in triplicate.
Briefly, the ELISA plate was coated with HGF (0.2 μg/ml, 100 µl/well, R&D Systems, Abingdon, United Kingdom) overnight at 4°C. Glypican-3 (R&D Systems) binding was quantified after 30 min over a range of concentrations in the absence (vehicle) or presence of a range of concentrations of ILB®. After three washes with PBS, streptavidin-HRP (according to manufacturer’s recommendations for the batch used, R&D Systems) was added to the wells for 1 h to label the bound Glypican-3. After a further 3 washes, the remaining HRP activity was quantified using an HRP substrate (DY999, R&D Systems) at 450 and 540 nm, with Glypican-3 levels determined by reference to a standard calibration curve. All conditions were assayed in triplicate.
4
Quantification of Recombinant BDNF
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Filtered (centrifree ultrafiltration device, Merck Group, Darmstadt, Germany) stock solutions of carrier free recombinant human BDNF (248-N4-250/CF, R&D Systems, Canada) of known concentrations (typically 250 mgL−1) in the phosphate buffered saline (PBS) pH 7.4 ± 0.2, 0.15 M (Biomed, Lublin, Poland) were prepared to remove aggregates and provide constant, free form protein molecules concentration in the solvent. To minimize errors in concentration measurements, two complementary spectrophotometric techniques were used: the BCA (protein quantification bicinchoninic acid assay, kit for low concentration, ABCAm, Cambridge, UK) and UV absorbance at 280 nm measured with microplate spectrophotometer (BioTek Epoch, United States). Prior to each measurement, the stock solution was diluted to a desired bulk concentration, typically 0.01–2 mgL−1. The exact concentration of these solutions after membrane filtration was determined by commercially available Enzyme-Linked Immunosorbent Assay (ELISA) (DY992, DY990, DY994, DY999, DY995, WA126, DY006, DY268, R&D Systems). The temperature of experiments was kept at a constant value equal to 298 ± 0.1 K.
5
Quantitative FNIII12-14 Domain Assay
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After coating with FN, a monoclonal antibody for the FNIII12–14 domain (also known as heparin II domain) was used (Santa Cruz Biotechnology, sc-18827; 1:30, 2 hours at 37°C). Samples were washed three times with DPBS/0.5% Tween 20. An anti-mouse IgG horseradish peroxidase–conjugated antibody (Invitrogen, 626520; 1:2000, 1 hour at RT) was then used. After the samples were washed twice, they were exposed to the substrate solution (R&D Systems, DY999) and incubated for 20 min at RT in the dark. A stop solution (R&D Systems, DY994) was added before the absorbance was read at 450 nm.
6
Kinetic Analysis of Biotinylated Nanobody Binding
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Binding of biotinylated NbB6-ABD to TNF and HSA was determined by ELISA, using streptavidin-HRP (DY998, Biotechne R&D Systems, USA) for detection. Plate wells (2592, Corning, USA) were coated with 1µg of TNF or HSA in carbonate buffer pH 9.6 overnight at 4 °C, then washed three times with 0.3 mL of PBS 1X 0.1% Tween 20 (PBST) and blocked with 3% BSA or 5% milk in 1X PBS for 1 h at room temperature. Wells were then washed three times with 0.3 mL PBST and incubated for 1h at RT with 100 µL of different concentrations of the biotinylated protein. Wells were again washed three times with 0.3 mL PBST and incubated for 1 h at RT with 100 µL streptavidin-HRP (1:200, DY998, Biotechne R&D Systems, USA). They were then washed four times with 0.3 mL PBST, revealed with 100 µL of 3,3′,5,5,5′-Tetramethylbenzidine (TMB) (DY999, Biotechne R&D Systems, USA), and stopped with 50 µL of 2N H2SO4. Binding signals were read at 450 nm in a plate reader (BOECO, Germany). KD estimation was carried out followed the method and fitting function described in [35 (link)]. Linear regression analysis using this function was performed using the MyCurveFit web server (https://mycurvefit.com/ , last accessed on 20 March 2023).
7
Lipigenine Stimulates HBD-2 Production
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EXAMPLE 5
Lipigenine™ was tested for its ability to stimulate an increase in HBD-2 concentration. The HBD-2 standard ABTS ELISA development kits were obtained from PeproTech (Cat# 900-K172). ELISA was performed according to the manufactory instructions of each kit by adding 100 μl/well of culture medium after overnight treatment. The substrate of ELISA reaction was using the substrate reagent from R&D Systems (DY999), and the reactions were stopped by adding 50 μl of 1N H2SO4 in each well. The Lipigenine™ culture was compared to the control medium which contained no other ingredients. The results were measured using a colorimeter, absorbance was measured at 450 nanometers (nm) within 30 minutes. Wavelength correction was set to 570 nm. The concentration of each sample was calculated using ELISA standard curve. [000126] The addition of Lipigenine™ showed increased HBD-2 concentrations at both 0.1% and 1% Lipigenine™ in solution as compared to the control. An increase in HBD-2 concentration of 7% was observed for a 0.1% Lipigenine™ formulation while an increase in HBD-2 expression of 23% was observed for a 1% Lipigenine™ formulation. These results are shown in
8
Lipigenine™ Stimulates HBD-1 Production
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Example 4
Lipigenine™ was tested for its ability to stimulate an increase in HBD-1 concentration. The HBD-1 standard AB TS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) ELISA development kits were obtained from PeproTech (Cat #900-K202). ELISA were performed according to the manufactory instructions of each kit by adding 100 μl/well of culture medium after overnight treatment. The substrate of ELISA reaction was using the substrate reagent from R&D Systems (DY999), and the reactions were stopped by adding 50 μl of 1N H2SO4 in each well. The Lipigenine™ culture was compared to the control medium which contained no other ingredients. The results were measured using a colorimeter, absorbance was measured at 450 nanometers (nm) within 30 minutes. Wavelength correction was set to 570 nm. The concentration of each sample was calculated using ELISA standard curve.
The addition of Lipigenine™ showed high HBD-1 concentration at both 0.1% and 1% Lipigenine™ in solution as compared to the control. An increase in HBD-1 concentration of 20% was observed for 0.1% Lipigenine™ while an increase in HBD-1 concentration of 95% was observed for 1% Lipigenine™. These results are shown in
9
Quantification of gp120-specific Antibodies by ELISA
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gp120-specific antibodies were measured by ELISA. Plates (Nunc maxisorp, flat bottom, Life Technologies; catalog #44240421) were coated with 2.5 μg/mL of gp120 protein from strain CN54 (Acro Biosystems; catalog #GP4-V15227) overnight. The plate was washed with PBS-Tween20 and blocked with SuperBlock™ Blocking Buffer (Thermo Scientific; catalog #37515), and bulk IgG was added. Unbound IgG antibodies were washed, and bound IgG antibodies were detected using HRP-conjugated anti-human IgG antibody. After another wash, the plates were developed with TMB substrate (R&D; catalog #DY999) for 5–10 minutes in the dark, and the reaction was stopped using stop solution (R&D; catalog #DY994). The plates were immediately read at an optical density of 450nm.
10
ELISA for F8 Phage Antibody Detection
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MaxiSorp flat-bottom 96-well plates (Nunc, Thermo Scientific, Europe) were covered overnight with F8 phage (5 × 109 pfu/ml). Subsequently, wells were washed with PBS and blocked with 1% albumin. Diluted serum (1/100 in PBS) was added (100 μl of diluted serum per well). The plate was incubated at 37 °C for 2 hours and washed with 0.05% Tween 20 in PBS (Serva, Europe) 5 times. Diluted detection secondary antibody was applied (100 μl per well): peroxidase-conjugated AffiniPure goat anti-mouse IgM (Jackson ImmunoResearch Laboratories) or peroxidase-conjugated AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch Laboratories). The plate was incubated for 1 h at room temperature in the dark. DuoSet of substrate reagents for peroxidase was used according to the manufacturer’s instructions (DY999, R&D Systems, Europe) and incubated for 20 minutes. Twenty-five μl of 2 N H2SO4 was added, and absorbance was measured at 450 nm (main reading) and 570 nm (background).
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