Organic chemicals and solvents, glyphosate standard and structurally similar compounds, and salts for buffers were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). The purity of standard solutions was ≥98%. The glyphosate primary antibody produced in chicken was purchased from Agrisera (Vannas, Sweden). The goat anti-chicken immunoglobulin conjugated to horseradish peroxidase (HRP) as a secondary antibody was obtained from TS Labor (Budapest, Hungary). Immunoassays were carried out in polystyrene γ-irradiated by SER-TAIN™ process sterile 96-well Costar HB microplates (Corning Inc., Corning, NY, USA) for colorimetric assay, and in low profile 96-well microplates with white wells for increased fluorescence (Bio-Rad Laboratories, Hercules, CA, USA). A QuantaRed Enhanced Chemifluorescent HRP Substrate Kit was used as the last step in the immunoassays (ThermoFisher Scientific Inc., Waltman, MA, USA).
Quantared enhanced chemifluorescent hrp substrate kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantaRed Enhanced Chemifluorescent HRP Substrate kit is a reagent designed for the detection of horseradish peroxidase (HRP) in immunoassays. The kit provides a chemifluorescent substrate that produces a fluorescent signal upon reaction with HRP, allowing for sensitive and quantitative detection of the enzyme.
Automatically generated - may contain errors
Lab products found in correlation
Black 96 well plate, by Thermo Fisher Scientific (2 mentions)
Perfect block, by MoBiTec (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Salts for buffers, by Merck Group (1 mentions)
Resorufin, by Merck Group (1 mentions)
Amplex red, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Biosesang (1 mentions)
Biotin coated plate, by Thermo Fisher Scientific (1 mentions)
Human aβ42 elisa kit, by Thermo Fisher Scientific (1 mentions)
Pooled human plasma apheresis derived, by Innovative Research (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Human serum from human male ab plasma, by Merck Group (1 mentions)
Sodium carbonate, by Merck Group (1 mentions)
Akta pure 25 system, by Cytiva (1 mentions)
Histrap hp column, by Cytiva (1 mentions)
Infinite f500 plate reader, by Tecan (1 mentions)
Hrp conjugated anti his tag antibody, by Abcam (1 mentions)
Igg1 fc, by R&D Systems (1 mentions)
Her2 fc, by R&D Systems (1 mentions)
8 protocols using quantared enhanced chemifluorescent hrp substrate kit
1
Glyphosate Immunoassay Protocol
2
Amyloid-β Quantification Assay Protocol
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ADHP (10-acetyl 3,7-dihydroxyphenoxazine, Amplex Red) was purchased at Invitrogen (Carlsbad, CA, USA). Hydrogen peroxide was obtained from a Quanta Red Enhanced Chemifluorescent HRP Substrate kit (Thermo Scientific, Waltham, MA, USA). Resorufin (424455) and phosphate buffered saline (PBS) was purchased from Sigma Aldrich (St. Louis, MO, USA) and Welgene (Gyeongsangbuk-do, Korea) respectively. PBS of various pH was purchased from the Biosesang (Seongnam-si, Gyeonggi-do, Korea). Biotin-coated plate and HRP-conjugated streptavidin were purchased from Thermo Scientific. Human Aβ42 ELISA kits were purchased from Invitrogen. Pooled Human Plasma Apheresis Derived was purchased from Innovative Research (Novi, MI, USA).
3
Quantitative Analysis of Biotin-HSA Nanowires
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The biotinylated HSA nanowires on a Si substrate (1.5 × 0.7 cm2) were incubated with 100 ng cm−3 HRP–streptavidin in 10 mM potassium phosphate buffer containing 0.15 M sodium chloride (pH 7.0) at 25 °C for 30 min, then the nanowires were washed with the same buffer three times. A QuantaRed Enhanced Chemifluorescent HRP Substrate kit (Thermo Scientific) was used for assaying the HRP activity26 (link). The nanowires was dipped into a mixture of 1.8 cm3 of 0.1 M potassium phosphate buffer (pH 7.0) and 0.2 cm3 of substrate solution containing 10-acetyl 3,7-dihydroxyphenoxazine and hydrogen peroxide. The enzymatic reaction mixture was stirred at 800 r.p.m. The time course of the enzymatic reaction was monitored continuously by measuring the fluorescence at 585 nm (excitation at 570 nm).
4
Microfluidic Malaria Detection Assay
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The microfluidic microplate plate (Optimiser™ microplate), holder, and absorbent pad were provided from MiCo BioMed Co., Ltd. (Seoul, Korea). Mouse monoclonal PfLDH antibody was purchased from Fapon Biotech (BRCMALS212, Guangdong, China), and rabbit polyclonal PfLDH antibody was purchased from LifeSpan BioScience, Inc. (LS-C488775, Seattle, USA). PfLDH and PvLDH were provided by BioNano Health Guard Research Center (H-GUARD). Polyclonal anti-rabbit HRP-tagged antibody (HRP-Ab) was obtained from Cell Signaling Technology, Inc. (Danvers, USA). Phosphate-buffered saline (PBS), blocking solution (StartingBlock™) and HRP substrate kit (QuantaRed™ Enhanced Chemifluorescent HRP substrate kit) were purchased from Thermo Fisher Scientific Ltd. (Waltham, USA). Bovine serum albumin (BSA) and sodium carbonate were purchased from Sigma-Aldrich (Louis, USA). Human serum (from human male AB plasma, USA origin, sterile-filtered) was purchased from Sigma-Aldrich (Louis, USA).
5
Purification and Characterization of scFv Protein
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HeLa cells (1 × 106 cells in 10-cm dish) were transfected with the pcDNA/ss-scFv vector using PEI. After incubation in 0.5% FBS-DMEM for 24 h, the secreted His6-tagged scFv protein in the culture medium was purified using a HisTrap HP column (Cytiva, Marlborough, MA, USA) with the AKTA pure 25 system (Cytiva).
ELISAs were carried out in 96-well black plates (Thermo Fisher Scientific) with all steps performed at room temperature with the exception of coating that was conducted by incubation with recombinant human CD25-Fc (R&D systems, Minneapolis, MN, USA) overnight at 4 °C. Blocking was performed with 2% Perfect Block (MoBiTec) in PBS (PBS-PB) for 2 h, followed by washing three times with PBS-T and incubation with His6-tagged scFv in PBS-PB for 1 h. The wells were then washed again three times with PBS-T and incubated with a 1000-fold diluted HRP-conjugated α-His tag antibody (Abcam, Cambridge, MA, USA) in PBS-PB for 1 h. Before detection using a QuantaRed Enhanced Chemifluorescent HRP Substrate kit (Thermo Fisher Scientific), the wells were washed three times with PBS-T and then another three times with PBS. The resulting fluorescence signals were measured using an Infinite F500 plate reader (Tecan, Mannedorf, Switzerland) with specific filters (Ex/Em = 535 nm/590 nm).
ELISAs were carried out in 96-well black plates (Thermo Fisher Scientific) with all steps performed at room temperature with the exception of coating that was conducted by incubation with recombinant human CD25-Fc (R&D systems, Minneapolis, MN, USA) overnight at 4 °C. Blocking was performed with 2% Perfect Block (MoBiTec) in PBS (PBS-PB) for 2 h, followed by washing three times with PBS-T and incubation with His6-tagged scFv in PBS-PB for 1 h. The wells were then washed again three times with PBS-T and incubated with a 1000-fold diluted HRP-conjugated α-His tag antibody (Abcam, Cambridge, MA, USA) in PBS-PB for 1 h. Before detection using a QuantaRed Enhanced Chemifluorescent HRP Substrate kit (Thermo Fisher Scientific), the wells were washed three times with PBS-T and then another three times with PBS. The resulting fluorescence signals were measured using an Infinite F500 plate reader (Tecan, Mannedorf, Switzerland) with specific filters (Ex/Em = 535 nm/590 nm).
6
Quantitative ADP-ribosylation Visualization
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Remove the Strep-HRP and wash the plate 3× with 100 μL 1× PBST and 1× with 100 μL 1× PBS, leaving each wash for ≥2 min at RT.
Prepare the QuantaRed solution per the instructions in the kit.
Add 100 μL QuantaRed solution to each well and incubate for 30 s to 1 min then quench with the QuantaRed stop solution.
7
Phage-ELISA and Indirect ELISA for HER2 Detection
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For phage-ELISA, the wells of 96-well black plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 1000 ng of HER2-Fc, streptavidin (Wako, Osaka, Japan), or IgG1-Fc (R&D systems) and blocked with 2% Perfect-Block (MoBiTec, Göttingen, Germany) in PBS for 1 h. Phages (1 × 1010 pfu) were added to each well and incubated for 1 h. After washing the plate, the bound phages were detected with the anti-T7 fiber tail antibody (Merck Millipore) as a primary antibody and the anti-mouse IgG HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA).
For indirect ELISA, the wells were first coated with 50 ng of HER2-Fc and then blocked with 2% Perfect-Block in PBS for 2 h. After incubation with sample proteins for 1 h, the HRP-conjugated anti-His-tag antibody (Abcam, Cambridge, MA, USA) was incubated for 1 h with the samples and then treated with the QuantaRed Enhanced Chemifluorescent HRP Substrate kit (Thermo Fisher Scientific). The resulting fluorescence was measured using an Infinite F500 (Tecan, Mannedorf, Switzerland) with specific filters (Ex/Em = 535 nm/590 nm).
For specificity evaluation, the wells were incubated overnight with HER2-Fc (50 ng/50 μL in PBS), EGFR-Fc (50 ng/50 μL in PBS; R&D systems), or 50 μL PBS, and applied to the indirect ELISA described above.
For indirect ELISA, the wells were first coated with 50 ng of HER2-Fc and then blocked with 2% Perfect-Block in PBS for 2 h. After incubation with sample proteins for 1 h, the HRP-conjugated anti-His-tag antibody (Abcam, Cambridge, MA, USA) was incubated for 1 h with the samples and then treated with the QuantaRed Enhanced Chemifluorescent HRP Substrate kit (Thermo Fisher Scientific). The resulting fluorescence was measured using an Infinite F500 (Tecan, Mannedorf, Switzerland) with specific filters (Ex/Em = 535 nm/590 nm).
For specificity evaluation, the wells were incubated overnight with HER2-Fc (50 ng/50 μL in PBS), EGFR-Fc (50 ng/50 μL in PBS; R&D systems), or 50 μL PBS, and applied to the indirect ELISA described above.
8
Biotinylation and Detection Reagents
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HSA, BSA, and OVA from egg were purchased from Sigma-Aldrich. Avidin from chicken egg white was purchased from Calzyme Laboratories. Trypsin from porcine pancreas was purchased from Nacalai Tesque. Biotin N-hydroxysuccinimide ester (NHS-biotin), HRP conjugated with streptavidin (HRP–streptavidin) and QuantaRed Enhanced Chemifluorescent HRP Substrate kit were purchased from Thermo Fisher Scientific. Streptavidin conjugated with Alexa Fluor 488 was purchased from Life Technologies. D -Biotin was purchased from AnaSpec. 3,6-Dioxaoctane-1,8-N-dibiotinamide (dibiotinyl linker) was prepared using literature methods from D -biotin27 . All other reagents and solvents were purchased from Wako Pure Chemical.
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