Abts solution

Manufactured by Merck Group

Sourced in United States, Germany, Italy

ABTS solution is a substrate for enzymatic assays, commonly used in colorimetric detection methods. It provides a chromogenic reaction that can be measured spectrophotometrically.

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Lab products found in correlation

Hrp conjugated anti mouse igg h l, by Koma Biotech (2 mentions) Hrp conjugated anti murine igg, by Thermo Fisher Scientific (2 mentions) Trolox, by Merck Group (2 mentions) Spectramax plate reader, by Molecular Devices (2 mentions) Hrp conjugated goat anti mouse igg antibody, by Jackson ImmunoResearch (1 mentions) Uv 1800, by Shimadzu (1 mentions) Hrp conjugated anti mouse iga, by Merck Group (1 mentions) Synergy ht multi mode microplate reader, by Agilent Technologies (1 mentions) Model 680 microplate reader, by Bio-Rad (1 mentions) 2 2 diphenyl 1 picrylhydrazyl (dpph), by Thermo Fisher Scientific (1 mentions) Cell death detection elisa kit, by Merck Group (1 mentions) Synergy multi mode reader, by Agilent Technologies (1 mentions) 3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions) Sunrise absorbance reader, by Tecan (1 mentions) Multiskan ex, by Thermo Fisher Scientific (1 mentions) Multiskan go, by Thermo Fisher Scientific (1 mentions) Potassium persulfate, by Merck Group (1 mentions) 96 well microplate, by Corning (1 mentions) Phenol red free dmem, by Thermo Fisher Scientific (1 mentions) Hrp conjugated goat anti mouse secondary antibody, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

ABTS) radical solution (3 citations) ABTS TM (2 citations) ABTS working solution (2 citations) (ABTS) substrate solution (2 citations)

25 protocols using abts solution

Quantification of GP-specific IgG Subclasses

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Each well of a 96 well plate was coated at 4℃ overnight with recombinant GP and GP1 proteins (0.5 µg/mL in PBS). The next day the plate was washed 3 times with washing buffer (PBS+0.05% Tween 20) and then blocked for 1 hour with blocking buffer (washing buffer+2% bovine serum albumin). Then, the plate was reacted with test samples for 1 hour. After this, the plates were washed 3 times with washing buffer and reacted for 1 hour with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (H [heavy]+L [light]) diluted in blocking buffer. After washing the plate, an ABTS solution (Merck Millipore, Billerica, MA, USA) was added to each well. Optical density (OD) was read at 405 nm using an ELISA reader. In particular, for the determination of the relative levels of GP-specific IgG subclasses, HRP-conjugated anti-murine IgG1, IgG2a, IgG2b and IgG3 antibodies (Thermo Scientific, Waltham, MA, USA) were substituted for HRP-conjugated anti-murine IgG (H+L). HRP-conjugated anti-mouse IgG (H+L) was purchased from Komabiotech (Seoul, Korea).

Han B.S., Jang H.Y., Racine T., Qiu X, & Sin J.I. (2018). Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein. Clinical and Experimental Vaccine Research, 7(2), 119-128.

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Binding Affinity of VLP Selectants

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ELISA was used to assess relative binding of VLP selectants to AP4-24H11. Briefly, wells were coated with 250 ng of purified VLPs in PBS and incubated overnight at 4°C. Wells were washed 3 times with PBS and blocked for 1 hour using 3% BSA. Different concentrations of AP4-24H11 in 3% BSA were applied to each well, and incubated at room temperature for 1 hour. Unbound antibody was removed by washing with PBS. Goat anti-Mouse IgG HRP conjugated antibody (Jackson ImmunoResearch Laboratories, West Grove PA) was diluted 1∶5000 in 3% BSA and incubated for 1 hour at room temperature. ABTS solution (EMD Millipore, Billerica MA) was used to detect bound HRP antibody and color change was measured by absorbance at 405 nm (Opsys Plate Reader, Thermo Scientific, Waltham MA).
For competition ELISAs, plates were prepared as above. AP4-24H11 (100 ng/well) was mixed with different concentration of the cyclical, bioactive AIP4 peptide (10 µM–0.1 µM) for 10 minutes prior to incubation with VLPs. Secondary antibody and detection was the same as above. As control peptides, we used a linear form of AIP4 or a cyclical peptide from the L2 protein of human papillomavirus 16.

O’Rourke J.P., Daly S.M., Triplett K.D., Peabody D., Chackerian B, & Hall P.R. (2014). Development of a Mimotope Vaccine Targeting the Staphylococcus aureus Quorum Sensing Pathway. PLoS ONE, 9(11), e111198.

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SARS-CoV-2 Antibody Detection Assay

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Receptor-binding domain (RBD) protein (1 μg/mL, 50 μL/well) (ACRO Biosystems, Newark, DE, USA) of the SARS-CoV-2 Wuhan strain or nucleocapsid (N) protein (1 μg/mL, 50 μL/well) (ACRO Biosystems, Newark, DE, USA) was coated onto enzyme-linked immunosorbent assay plates overnight at 4 °C. The plates were washed with phosphate-buffered saline containing 0.1 % Tween 20 (PBST) and incubated with 20 % Blocking-One plus PBST. After blocking, serum diluted 400- or 1600-fold for RBD protein, and 100-fold for N protein was added, and the cells were washed. Next, anti-human IgG horseradish peroxidase (Promega, Madison, WI, USA) was added, washed, and ABTS solution (Merck, Darmstadt, Germany) was added. After stopping the reaction with 4 N sulfuric acid, the enzyme activity was detected at 405/490 nm using a plate reader. An absorbance of 1.0 or higher was interpreted as positive for anti-N protein IgG. The dynamic ranges were 1 to 1.0 × 10⁶ for neutralizing antibody titers, and 0 to 8.0 × 10⁴ U/mL for anti-RBD IgG and anti-N protein IgG, respectively.

Itamochi M., Yazawa S., Inasaki N., Saga Y., Yamazaki E., Shimada T., Tamura K., Maenishi E., Isobe J., Nakamura M., Takaoka M., Sasajima H., Kawashiri C., Tani H, & Oishi K. (2023). Neutralization of Omicron subvariants BA.1 and BA.5 by a booster dose of COVID-19 mRNA vaccine in a Japanese nursing home cohort. Vaccine, 41(13), 2234-2242.

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Evaluation of Sidr Honey's Antioxidant Capacity

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In accordance with Re et al. [42 (link)], Sidr honey was evaluated for its ABTS free radical scavenging activity. As a first step, distilled water (dH₂O) was used to prepare an ABTS solution (7 mM) (Merck KGaA, Darmstadt, Germany). Following the preparation of the ABTS, the ABTS was mixed with potassium persulfate (2.454 mM) to form ABTS (radical cation). Before using the generated ABTS, it was placed at room temperature in the dark for 12–16 h. The prepared ABTS was further diluted with dH₂O until the absorbance was at a point of 0.70 (at 734 nm) (UV-1800, Shimadzu, Japan). Then, the honey sample (0.07 mL) and the ABTS (3 mL) were mixed together and incubated at room temperature for 7 min in the dark and the absorbance of the mixture was measured using a spectrophotometer at 734 nm. To calculate antioxidant activity, the following equation was used:

% Inhibition = \frac{A_{c o n t r o l} - A_{s a m p l e}}{A_{c o n t r o l}} \times 100

where A_control = absorbance of negative control at the moment of solution preparation and A_sample = absorbance of sample after incubation.

Bazaid A.S., Alsolami A., Patel M., Khateb A.M., Aldarhami A., Snoussi M., Almusheet S.M, & Qanash H. (2023). Antibiofilm, Antimicrobial, Anti-Quorum Sensing, and Antioxidant Activities of Saudi Sidr Honey: In Vitro and Molecular Docking Studies. Pharmaceutics, 15(9), 2177.

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Quantifying Anti-GP Antibody Responses

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Each well of a 96 well plate was coated at 4℃ overnight with recombinant GP (0.5 µg/mL in PBS). The next day, the plate was washed 3 times with washing buffer (PBS+0.05% Tween 20) and blocked for 1 hour with blocking buffer (washing buffer +2% bovine serum albumin). Then, the plate was reacted for 1 hour with horseradish peroxidase (HRP)–conjugated anti-mouse IgG (H+L), HRP-conjugated anti-mouse IgM, HRP-conjugated anti-mouse IgA, or HRP-conjugated anti-mouse IgG (Fc-specific) diluted in blocking buffer. After washing the plate, an ABTS solution (Merck Millipore, Billerica, MA, USA) was added to each well. The optical density was read at 405 nm using an ELISA reader. For the determination of the relative levels of GP-specific IgG subclasses, we substituted HRP-conjugated anti-murine IgG1, IgG2a, IgG2b, and IgG3 Abs (Thermo Scientific) for HRP-conjugated anti-murine IgG (H+L). HRP-conjugated anti-mouse IgG (H+L) and HRP-conjugated anti-mouse IgM were purchased from Komabiotech (Seoul, Korea). HRP-conjugated anti-mouse IgG (Fc-specific) and HRP-conjugated anti-mouse IgA were purchased from Sigma-Aldrich and Thermo Scientific, respectively.

Lee S.H., Han B.S., Choe J, & Sin J.I. (2017). Preferential production of IgM-secreting hybridomas by immunization with DNA vaccines coding for Ebola virus glycoprotein: use of protein boosting for IgG-secreting hybridoma production. Clinical and Experimental Vaccine Research, 6(2), 135-145.

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ABTS Radical Scavenging Assay for Antioxidant Evaluation

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The TEAC assay is based on the reduction of the 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical induced by antioxidants. The ABTS radical cation (ABTS^+● was prepared by mixing a 7 mM ABTS solution (Sigma-Aldrich, Milan, Italy) with 2.45 mM potassium persulfate (1:1) and stored for 16 h at room temperature and in dark. To prepare the ABTS reagent, the ABTS^+● was diluted in 5 mM phosphate buffer (pH 7.4) to obtain a stable absorbance of 0.700 (±0.02) at 730 nm. For the assay, 10 µL of BUO extract (at the final concentrations of 0.5, 1.0, 5.0, and 10.0 µg/mL) were added to 140 µL of diluted the ABTS^+●. The microplate was incubated for 30 min at 30 °C and the absorbance was read at 730 nm using a Synergy™ HT-multimode microplate reader (Biotek Instruments, Winooski, VT, USA). The TEAC values were calculated using a Trolox (Sigma-Aldrich, Milan, Italy) calibration curve (60–320 µM) (Figure S1).

Bartolomei M., Bollati C., Bellumori M., Cecchi L., Cruz-Chamorro I., Santos-Sánchez G., Ranaldi G., Ferruzza S., Sambuy Y., Arnoldi A., Mulinacci N, & Lammi C. (2021). Extra Virgin Olive Oil Phenolic Extract on Human Hepatic HepG2 and Intestinal Caco-2 Cells: Assessment of the Antioxidant Activity and Intestinal Trans-Epithelial Transport. Antioxidants, 10(1), 118.

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Antioxidant Activity Evaluation Using ABTS and DPPH

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Spectroscopic methods were used to measure the scavenging activities of the sample against 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) cation radical (ABTS^•⁺) and 2,2-diphenyl-1-picrylhydrazyl radial (DPPH^•) [31 (link),32 (link),33 (link)]. The ABTS^•⁺ solution was prepared by mixing 0.54 mM ABTS solution (Sigma-Aldrich) and 0.27 mM potassium persulfate solution (Sigma-Aldrich) in equal volumes and allowing them to react for 24 h at room temperature (25 °C) in the dark. Each serial dilution of a plant-derived material or compound in ethanol (100 μL) was reacted with 0.27 mM ABTS^•⁺ in water (100 μL) of at 25 °C for 3 min, followed by measurement of the absorbance at 734 nm with a BioRad Model 680 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For the DPPH^• scavenging activity assay, a serially diluted sample in ethanol (100 μL) was mixed with 0.2 mM DPPH^• (Alfa Aesar, Ward Hill, MA, USA) in ethanol and reacted at 25 °C for 30 min. The absorbance was measured at 517 nm using a microplate reader.

Bae I.A., Ha J.W., Choi J.Y, & Boo Y.C. (2022). Antioxidant Effects of Korean Propolis in HaCaT Keratinocytes Exposed to Particulate Matter 10. Antioxidants, 11(4), 781.

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Quantifying NET Release and MPO-DNA Complexes

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NETs release in culture and plasma was quantified with Quanti-IT Pico
Green double stranded (ds) DNA kits (Thermo Fisher Scientific) using lambda DNA
as quantitation standards in accordance with manufacturers recommendations. To
capture NET fragments from BALF 100 μg of rabbit polyclonal Chip grade
Abs specific for citrullinated histone H3 (Abcam) or 500 μg control
rabbit polyclonal Abs (Jackson Labs) were conjugated to 1.7 mg of 0.3 mm
diameter IDC UltraClean Amidine Latex beads (Fisher Thermo Scientific) in
accordance with manufacturers recommendations. BALF was split into two 1.5 ml
aliquots and incubated with either 250 μg H3 citrullinated Ab-beads or
control Ab-beads at 4 °C for 3 hours. Beads were then washed twice in
cold PBS and directly quantified for dsDNA content with Pico Green relative to
dsDNA content adsorbed to control Ab-beads. MPO-DNA complex detection in
circulating plasma was conducted with MPO ELISA kit plates (ThermoFisher
Scientific) precoated with MPO capture Abs. Then following plasma incubation for
3 hours at 4° C and 3 washes of cold ELISA washer buffer plates were
incubated with anti-DNA peroxidase conjugated Abs from a Cell Death Detection
ELISA kit (Sigma), washed three times, and incubated with ABTS solution (Sigma)
for 20 minutes. Complexes were quantified as optical density at 405 nm using a
Synergy Multimode reader (Biotek).

Scozzi D., Wang X., Liao F., Liu Z., Zhu J., Pugh K., Ibrahim M., Hsiao H.M., Miller M.J., Yizhan G., Mohanakumar T., Krupnick A.S., Kreisel D, & Gelman A.E. (2018). Neutrophil extracellular trap fragments stimulate innate immune responses that prevent lung transplant tolerance. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 19(4), 1011-1023.

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ABTS Radical Scavenging Assay for Antioxidant Capacity

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The antioxidant capacity assay was carried out by the improved ABTS^•+ spectrophotometric method [67 (link)]. In this method, ABTS^•+ solution (Sigma Aldrich, St. Louis, MO, USA) was mixed with different amounts of the extractive solution obtained from N. tabacum inflorescence powder. Conventional and non-conventional solvent controls were performed in each determination to scan any possible interference. Absorbance was recorded at 734 nm after 6 min. Results are expressed as SC₅₀ values. SC₅₀ (μg GAE/mL) was defined as the concentration of phenolic compounds necessary to scavenge the 50% of ABTS free radicals. Quercetin was used as a reference compound.

Leal M., Moreno M.A., Albornoz P.L., Mercado M.I., Zampini I.C, & Isla M.I. (2023). Morphological Characterization of Nicotiana tabacum Inflorescences and Chemical-Functional Analysis of Extracts Obtained from Its Powder by Using Green Solvents (NaDESs). Plants, 12(7), 1554.

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Antibody Quantification by ELISA

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Specific IgE, total IgG, IgG1 and IgG2a antibodies were determined by in-house enzyme-linked immuno-sorbent assay (ELISA) with the required antibodies purchased from BD Pharmingen (San Jose, CA, USA.). Micro-titre plates were coated with midge extract for 2 hours at 37°C. After washing with PBST, the plates were blocked with 5% skimmed milk (for IgE) or 2% bovine serum albumin (BSA) (for all IgGs) for 2 h at room temperature. Sera were diluted (1∶10 for IgE or 1∶100 for all IgGs) in PBST and incubated at 4°C overnight (for IgE) or room temperature for 2 h (for all IgGs).
For IgE measurement, the plates were incubated with biotin-conjugated rat anti-mouse IgE (1∶4000) for 2 h at room temperature. Subsequently, the plates were reacted with horseradish peroxidase-conjugated streptavidin (1∶10,000) for 1 h, developed by adding TMB (Sigma), and stopped with 1 M H₃PO₄. For IgG measurement, the plates were incubated with horseradish peroxidase-conjugated rabbit anti-mouse IgG (1∶10,000), IgG1 (1∶5,000) or IgG2a (1∶1000) for 2 h at room temperature and developed by adding ABTS solution (Sigma). The optical density was then analyzed on a Sunrise Absorbance Reader (TECAN, Austria) at 450 nm and 415 nm, respectively.

Lee M.F., Yang K.J., Wang N.M., Chiu Y.T., Chen P.C, & Chen Y.H. (2014). The Development of a Murine Model for Forcipomyia taiwana (Biting Midge) Allergy. PLoS ONE, 9(3), e91871.

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