Sequencing libraries were prepared as described (Rouskin et al., 2014 (link)). Specifically, DMS treated mRNA samples were denatured for 2 min at 95°C and fragmented at 95°C for 2 min in 1x RNA fragmentation buffer (Zn2+ based, Ambion). The reaction was stopped by adding 1/10 vol of 10X Stop solution (Ambion) and quickly placed on ice. The fragmented RNA was run on a 10% TBU (Tris borate urea) gel for 60 min. Fragments of 60–70 nucleotides in size were visualized by blue light (Invitrogen, Carlsbad CA) and excised. Reverse transcription was performed in a 20 µL volume at 52°C using Superscript III (Invitrogen), and truncated reverse transcription products of 25–45 nucleotides were extracted by gel purification.
Stop solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
Stop Solution is a laboratory reagent used to halt or terminate a specific chemical reaction or process. It is commonly used in various analytical techniques, such as enzyme-linked immunosorbent assays (ELISAs), to stop the color development reaction and stabilize the results for analysis.
Automatically generated - may contain errors
Lab products found in correlation
Tmb substrate, by Thermo Fisher Scientific (6 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Thermo Fisher Scientific (4 mentions)
High binding elisa plate, by Corning (3 mentions)
Tmb substrate solution, by Thermo Fisher Scientific (3 mentions)
1 step ultra tmb substrate, by Thermo Fisher Scientific (3 mentions)
Goat polyclonal antibody to human igg, by Abcam (3 mentions)
Maxisorp plate, by Thermo Fisher Scientific (2 mentions)
Victor nivo, by PerkinElmer (2 mentions)
Maxisorp elisa plate, by Thermo Fisher Scientific (2 mentions)
Epoch 2 plate reader, by Agilent Technologies (2 mentions)
El406 plate washer dispenser, by Agilent Technologies (2 mentions)
One step tmb, by Thermo Fisher Scientific (2 mentions)
Avidin hrp, by Thermo Fisher Scientific (2 mentions)
Tmb substrate, by Bio-Rad (2 mentions)
High binding 96 well plate, by Corning (2 mentions)
Hrp conjugated anti human igg antibody, by Thermo Fisher Scientific (2 mentions)
Carbonate coating buffer, by Thermo Fisher Scientific (2 mentions)
Mage a4, by OriGene (2 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Novex 10 tbe urea gel, by Thermo Fisher Scientific (1 mentions)
43 protocols using stop solution
1
DMS-Seq Library Preparation Protocol
2
DMS-Seq Library Preparation and Fragmentation
3
Comparative Analysis of Leukemia and Breast Cancer Cell Lines
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The THP-1 cells are designates a spontaneously immortalized monocyte-like cell line, derived from the peripheral blood of a childhood case of acute monocytic leukemia, AMJ-13 cells are Iraqi patient breast cancer cell line it is a receptor-negative, hormone-nonresponsive, and HBL cells are breast epithelial cell line. All cells were kindly provided by the Iraqi Centre for Cancer and Medical Genetic Research (ICCMGR), Al-Mustansiriyah University, Baghdad, Iraq. RPMI-1640, trypsin-EDTA, dimethyl sulfoxide (DMSO), fetal bovine serum, ovalbumin, 3-(4,5-dimethylthiazal-z-yl)-2,5-diphenylterazolium (MTT), lipopolysaccharides (LPS) from Escherichia coli O111:B4 (Cat. No. L2630, Sigma- Aldrich, MO, USA), adenosine triphosphate (ATP), and Triton X-100 were from Sigma (MO, USA). 3,3′,5,5′-Tetramethylbenzidine (TMB) microwell peroxidase substrate solution was obtained from Kirkegaard & Perry Laboratories, Inc. (KPL) (Gaithersburg, MD, USA). Stop solution was obtained from eBioscience (Wembley, UK). The remaining chemicals and reagents were of analytical grade.
4
Profiling Autoantibody Levels in Serum
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Briefly, 96-well microtiter plates were coated with polyclonal anti-collagen (Abcam, Cambridge, UK), anti-filaggrin (Santa Cruz Biotechnology, Dallas, Texas, USA), or anti-fibronectin (Abcam) antibodies (1:100 dilution) in 100μl of coating buffer (eBioscience) and incubated overnight at 4°C. The plates were washed seven times with PBS-T and incubated with assay buffer (eBioscience) for 1 h at room temperature (RT). The plates were again washed seven times with PBS-T. Serum samples (1:10 diluted in PBS) were added to each well in triplicate, and the plates were incubated for 2 h at RT. The plates were washed seven times with PBS-T, and then the 12G1 mAb (1:100 diluted in assay buffer) was added, and the plates were incubated for 2 h at RT. After incubation, the plates were washed seven times in PBS-T, 50µl of HRP-conjugated anti-mouse IgG antibody (1:3000, Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK) was added, and the plates were incubated for a further 1 hour at 37 °C. Finally, the plates were washed nine times, and the bound antibodies were visualized by adding substrate solution (eBioscience). Reactions were stopped after 15 mins by adding 50µl of stop solution (eBioscience). The colorimetric reaction was measured at 450 nm on a VersaMax ELISA reader.
5
MERS-CoV Antibody Detection Assay
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The 96-well Maxisorp plates (Thermo Fisher Scientific) were coated with 100 ng of MERS-CoV nucleocapsid protein (NP; Sinobio, Beijing, China) and spike protein (S1 subunit; Sinobio) in PBS overnight at 4 °C. Plates were blocked with 100 μL Tris-buffered saline with 0.01% tween-20 (TBS-T) and 5% skim milk for 1 h at 37 °C. After washing with TBS-T, plates were incubated with serum (100 μL/well) diluted 3-fold at 1:1000 to 1:243,000 prepared in TBS-T with 3% skim milk for 1 h at 37 °C. Plates were washed and incubated with anti-mouse IgG/IgG2c/IgG1-HRP (Sigma-Aldrich) at 1:5000 dilutions in TBS-T with 3% skim milk for 1 h at 37 °C. Plates were developed using a tetramethyl benzidine (TMB; Thermo Fisher) substrate and stopped with a stop solution (Thermo Fisher). Absorbance was measured at 450 nm using a microplate reader (VICTOR Nivo™; PerkinElmer, Waltham, MA, USA).
6
ELISA-Based Anti-RSV IgG Quantification
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Sera were evaluated for anti-RSV IgG levels as described (51 (link)). Briefly, high-binding ELISA plates (Corning, Corning, NY) were coated with 5 µg/mL RSV A2 or B1 lysate overnight at 4°C. The next day, wells were washed 3x with KPL wash buffer (1x KPL in distilled water (diH2O) (SeraCare, Milford, MA) and blocked with Blotto (5% non-fat dry milk) overnight at 4°C. Blotto was removed and sera (in 3-fold dilutions starting at 1:50) was diluted in Blotto and added to wells overnight at 4°C. Wells were washed 3x with KPL wash buffer and 2° goat-anti-mouse IgG-HRP (ThermoFisher, Waltham, MA), or secondary subtype IgG1 or IgG2a antibodies (Southern Biotech, Birmingham, AL) were added. Plates were incubated overnight at 4°C, washed 3x with KPL wash buffer, and developed with 1-Step™ Ultra 3,3’,5,5’-tetramethylbenzidine (TMB; ThermoFisher) for 20 min, and stopped with Stop Solution (ThermoFisher), then read immediately using a BioTek plate reader (BioTek, Winooski, VT) at OD450.
7
SPARC Binding Assay for TLR Interactions
8
Quantifying DC IL-12p70 Production in Response to CD40L
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DC production of IL-12p70 in response to CD40L-transfected J558 cells (J558-CD40L; a gift from Dr. P. Lane, Birmingham, UK) stimulation was determined as previously described [7 (link)]. Briefly, DC were plated (2.5 × 104 cells/well) in a 96-well flat-bottom plate and stimulated with J558-CD40L (5 × 104 cells/well) for 24 h. Culture supernatants were collected and tested by IL-12p70 ELISA using the following reagents: Recombinant Human IL-12 Standard (R&D Systems Cat# 219-IL-005), Primary Human IL-12 mAb (Thermo Scientific Cat#M122), Secondary Human IL-12 mAb, Biotin-labeled (Thermo Scientific Cat# M121B), HRP-conjugated Streptavidin (Thermo Scientific Cat# N100), TMB Substrate Solution (Thermo Scientific Cat# N301), Stop Solution (Thermo Scientific Cat# N600).
9
Quantification of Collagen-specific Antibodies
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50ng/μl type II collagen were coated with 100 μl coating buffer (BioLegend, San Diego, CA, US) on Corning Costar Brand 96-Well EIA/RIA Plate (Thermo Fisher, Waltham, Massachusetts, US) overnight. After washing and blocking with 1% BSA buffer, mice sera or joint lysate (as mentioned in cytokine assay) were diluted from 1:10 to 1:107 with three times dilution and incubated for 2h at room temperature. After wash, HRP conjugated anti-mouse antibody (abcam, Cambridge, UK, 1/10000 dilution) was added in each well for 1h at room temperature, then TMB substrate (abcam, Cambridge, UK) was added for 15 min and reactions stopped with stop solution (Thermo Fisher, Waltham, Massachusetts, US), the color intensity was analyzed at 450nm, and the antibody titer was determined by the OD value that was 3 times higher than that in naïve mice.
10
ELISA for Spike Protein Detection
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We performed ELISAs as previously described [15 (link)]. Briefly, spike proteins were suspended at 1 μg/ml in 1 × PBS. One hundred microliters of protein suspension was added to each well of a 96-well Nunc MaxiSorp ELISA plate and allowed to coat overnight at 4 °C for 16 h. Wells were washed three times with 300 μl of 1× PBS + 0.05% Tween20 (wash buffer) followed by blocking for 2 h at room temperature with 200 μl of 1× PBS + 0.05% Tween20 + 5% Non-fat dry milk (blocking buffer). Wells were washed again three times with 300 μl of wash buffer prior to addition of 100 μl of sample diluted in blocking buffer (serum samples were heat inactivated for 45 min at 56 °C and diluted at 1:400 in blocking buffer). Samples were incubated for 1 h at room temperature, then washed three times with 300 μl of wash buffer. One hundred microliters of 1-Step Ultra TMB Substrate (ThermoFisher) was added and the plate was incubated for 10 min prior to stopping the reaction with 1 N sulfuric acid (Stop Solution, ThermoFisher). Absorbance was read at 450 nm and 650 nm on a BioTek Epoch2 plate reader. The process is semi-automated through the use of a BioTek EL406 plate washer/dispenser and two BioStack 4 plate stackers to minimize plate-to-plate variation and increase throughput (see Klumpp-Thomas C, Kalish H et al. 2020 for detailed automation methods) [15 (link)].
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