After cell lysis, protein samples were quantified and diluted as appropriate for the ELISA (~100 to 200 ng/ml protein). PathScan Phospho-Akt2 (Ser474) and Total Akt2 Sandwich ELISA kits were used to quantify AKT2 levels (Cell Signaling Technology, #7048 and #7046, respectively) as per the manufacturer’s instructions, by colorimetric reading at 450 nm on a Thermo Fisher Multiskan Go plate reader. Results are plotted as the OD450 (optical density at 450 nm) ratio of phosphorylated over total AKT2 or as fold change compared to the basal unstimulated state.
Multiskan go plate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Finland, France
The Multiskan GO plate reader is a versatile instrument designed for absorbance-based measurements in microplates. It enables a range of applications, including spectrophotometric analysis, enzyme-linked immunosorbent assays (ELISAs), and cell-based assays. The Multiskan GO provides reliable and accurate results across a variety of wavelengths and sample types.
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Lab products found in correlation
Hrp conjugated goat anti rabbit igg, by BD (2 mentions)
Cultrex bme, by Bio-Techne (2 mentions)
Celltiter 96 non radioactive cell proliferation assay, by Promega (2 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (2 mentions)
Cytotox 96 assay kit, by Promega (2 mentions)
Skanit sotware 3, by Thermo Fisher Scientific (2 mentions)
Skanit software, by Thermo Fisher Scientific (2 mentions)
Phenobooth imager, by Singer Instruments (2 mentions)
Pathscan phospho akt2 ser474, by Cell Signaling Technology (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
Tmb substrate, by Thermo Fisher Scientific (1 mentions)
Tetramethylbenzidine, by Thermo Fisher Scientific (1 mentions)
Nunc maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Bio-Rad (1 mentions)
Goat anti mouse igg1 antibody, by Southern Biotech (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
O phenylendiamine dihydrochloride opd, by Merck Group (1 mentions)
Hrp conjugated goat anti mouse igg, by BD (1 mentions)
84 protocols using multiskan go plate reader
1
Quantifying Phosphorylated and Total AKT2 Levels
2
Quantification of Intracellular ROS Levels
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Intracellular reactive oxygen species were quantified with Reactive Oxygen Species Assay Kit (Beyotime Institute of Biotechnology) as previously described.48 , 51 Briefly, cells were cultured with dihydrodichlorofluorescein diacetate (DCF‐DA) in serum‐free medium at 37˚C for 20 minutes and then added BaP (1.0 µmol/L) or/and Der f 1 (30 µg) in the presence or absence of NAC (1.0 µmol/L) for 1 hours. The DCF fluorescence was quantitated with a Multiskan Go plate reader (Thermo), with an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and normalized with the total protein content.
3
Enzyme-Linked Immunosorbent Assay for Anti-HSF1-PO4 Antibodies
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Phosphorylated and non-phosphorylated synthetic peptides or recombinant HSF1-PO4 protein (Abcam #115508) were diluted in carbonate/bicarbonate coating buffer (4 µg/mL) and incubated overnight in 96-well NUNC maxisorp plates at 4 °C. Plates were washed with wash buffer (PBS/0.05% v/v Tween-20) and blocked with PBS/1% BSA for a minimum of 1 h at 37 °C. After washing, plasma/sera were added (duplicate wells) and incubated at 4 °C overnight (1:10 or 1:20 dilution in wash buffer). After washing, HRP-conjugated goat anti-human IgA alpha chain (1:10,000, Abcam #98558) was added and incubated at 37 °C for 1.5 h. Following washing, the reaction was developed using TMB substrate (Invitrogen, Waltham, MA, USA) and stopped with 1 M HCl. Absorbance was immediately read at 450 nm (Optical Density (OD)450nm) using a Multiskan Go plate reader (Thermo Scientific, Waltham, MA, USA). Where specified, OD450nm values were normalised against values from a single patient sample consistently run across multiple experiments to account for inter-assay variability. The selected sample (OV0023, refer to Supplementary Table S2 ) had a mid-range anti-HSF1-PO4 or anti-peptide response (ranging OD450nm 0.41–1.0, depending on the target) and was thus chosen to avoid extreme measurements that could introduce bias.
4
Quantifying Alkaline Phosphatase Activity
5
Enzyme Kinetics Assay Protocol
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Carboxylic acids were dissolved to near saturation in assay buffer with 20% DMSO. Substrates were titrated in 1.7× dilutions over 8 points. Rates were measured continuously over 6 min in a ThermoFisher MultiSkan GO plate reader in precision mode. Rates were fitted to the Michaelis–Menten model by non-linear least-squares regression in GraphPad Prism v. 8.2.
6
Quantifying Allergic Responses: IgE, IgG1, and Mast Cell Mediators
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Total IgE was quantified in plasma, collected at day 35, using a total mouse IgE kit (BD Bioscience, Allschwil, Switzerland) according to the manufacturer’s protocol. For OVA-specific IgG1, 96-well Nunc MaxiSorp plates (Thermo Fischer Scientific, Zug, Switzerland) were coated with 100 μg/mL OVA (Sigma) overnight at 4°C. Wells were washed with PBS containing 0.05% Tween-20 (Bio-Rad, Reinach, Switzerland) and blocked with PBS-1% bovine serum albumin (Sigma) for 1 h at room temperature. Serially diluted standard monoclonal mouse anti-OVA IgG1 (used as a proxy for Th2 immune response) [37 (link)] (Antibody Shop; LucernaChem, Lucerne, Switzerland) and plasma samples were incubated for 2 h at 37 °C, followed by a 2-h incubation with an horseradish peroxidase (HRP)-labelled goat anti-mouse IgG1 antibody (1/5000; Southern Biotech, Allschwil, Switzerland). Plates were developed using tetramethylbenzidine (Thermo Fischer Scientific) and read at 450 nm with a Multiskan Go plate reader (Thermo Fisher Scientific).
Plasma mMCP-1, a mast cell-derived mediator of allergic reaction, was quantified using a mouse MCP-1 ELISA kit (Moredun Scientific, Penicuik, United Kingdom) according to the manufacturer’s instructions.
Plasma mMCP-1, a mast cell-derived mediator of allergic reaction, was quantified using a mouse MCP-1 ELISA kit (Moredun Scientific, Penicuik, United Kingdom) according to the manufacturer’s instructions.
7
UV-Vis Spectroscopy of Silk Fibroin
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The UV–vis absorption spectra of SF film before and after UV-irradiation were recorded with a Multiskan GO plate reader (Thermo Scientific, Waltham, MA, USA) in the range of 270 to 360 nm. Films were fixed inside of the cuvette cell. Absorbance of SF molecules released from SF films during immersion in 6 M urea solution at 10 mg/mL was also collected. The fluorescence intensity (excitation: 360/40 nm; emission: 460/40 nm) of the SF molecules in 6 M urea solution was measured by microplate reader (HT-Synergy, Bio-Tek, Winooski, VT, USA). For film and solution samples, the measured values of empty cuvette and the same volume of 6 M urea solution were used for blank subtraction, respectively.
8
C1q Binding Assay of rTs-Pmy
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To determine whether rTs-Pmy bound to complement C1q, plates were coated with different amounts of human C1q (0, 0.5, 1.0, 1.5 μg) and BSA (2 μg) in 100 μl of coating buffer (100 mM Na2CO3/NaHCO3, pH 9.6) at 4°C overnight. After washing three times with 1× phosphate buffered saline (PBS) pH 7.4 containing 0.05% Tween-20 (PBST), the plates were blocked with PBS containing 2% BSA for 2 h at 37°C. The different amounts of rTs-Pmy (0, 1, 2, 3, 4 μg) in 100 μl of 20 mM Tris-HCl, 50 mM NaCl and 1 mM CaCl2, pH 7.4 were added for 1 h at 37°C. The plates were washed three times with PBST, the binding of rTs-Pmy to human C1q was determined with anti-Ts-Pmy monoclonal antibody 9G3 (1:2,500 in 1% BSA/PBS). HRP-conjugated goat anti-mouse IgG (BD Biosciences, USA; 1:10,000 in 1% BSA/PBS) was used as the secondary antibody and o-phenylendiamine dihydrochloride (OPD, Sigma, USA) was used as the substrate. The absorbance of the supernatant was measured at 450 nm with a MultiskanGO plate reader (Thermo, USA).
9
PLA2 Inhibition Assay using 5-LOX
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The PLA2 inhibition assay was based on an indirect enzymatic method, which uses 5-LOX as coupling enzyme [38 (link)]. Briefly, 20 µL of PLA2 (1.75 µg/mL) and 20 µL of 5-LOX (1.61 µg/mL) in 3 mM deoxycholate dissolved in 50 mM Tris-HCL buffer (pH 8.5) were mixed with 50 µL of different concentrations of EnP(5,8) dissolved in the same buffer (3.125–50 µM). The reaction was initiated with the addition of 20 µL of DL-PC (PLA2 substrate) at 455 µM in 10 mM deoxycholate dissolved in 50 mM Tris-HCL buffer (pH 8.5). Then, the linoleic acid released through PLA2 activity was oxidized by 5-LOX at 37 °C, the increase in absorbance at 234 nm being followed spectrophotometrically in a Multiskan GO plate reader (Thermo Fisher Scientific; Waltham, MA, USA). Three independent experiments were performed in triplicate.
10
Preparation and Quantification of Ant Enzyme Extracts
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Whole bodies of S. invicta (10 large fire ant workers for EST and GST or 50 heads of large fire ant workers for AChE) were homogenized in ice-cold phosphate buffer (0.1 M, pH 7.0) in a Glass-Col homogenizer. The homogenate was centrifuged (4 °C, 12,000× g) in an Eppendorf microcentrifuge for 15 min and the supernatant fraction was then removed by filtration through glass fiber and collected for the following enzyme activity assays immediately. Total protein concentration of each enzyme extraction sample was measured by using a Bradford protein assay kit as described by using bovine serum albumin as a standard [20 (link)] (Thermo Scientific., Waltham, MA, USA). Protein content of the enzyme solution was quantified according to the method of Bradford (1976) [20 (link)], by using Coomassie Brilliant Blue G-250. Absorbance values were recorded 595 nm using a Thermo Scientific Multiskan Go plate reader.
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