Heparan sulfate proteoglycan-2 (HSPG2) deposition was assayed by semi-quantitative immunocytochemical detection. Cells were fixed in 10% neutral buffered formalin (Sigma), endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in methanol for 1 h, and samples blocked overnight in 3% goat serum. Samples were then incubated with monoclonal mouse anti-HSPG2 antibody (ab23418, Abcam, Waltham, MA) for 2 h followed by a series of washes with 1X PBS and labeling with a horseradish peroxidase-linked goat anti-mouse secondary antibody (polyclonal, 0.5 μg/mL; Sigma) for 40 min. After washing, samples were incubated in 1-Step Turbo-TMB ELISA substrate solution (ThermoFisher) and the reaction stopped after 5 min through the addition of 2 N sulfuric acid (ThermoFisher). Color development was proportional to HSPG2 content and measured with a plate reader. Background signal was determined by following the same procedure without incubating in primary antibody solution. This background absorbance was subtracted from the samples for the respective culture condition. Corrected absorbance values were normalized to total cell number determined by DAPI staining.
1 step turbo tmb elisa substrate solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 1-Step Turbo TMB-ELISA Substrate Solution is a ready-to-use reagent designed for use in enzyme-linked immunosorbent assay (ELISA) applications. It contains the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) and the necessary components to facilitate a colorimetric reaction, which can be used to quantify the presence of target analytes in a sample.
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Lab products found in correlation
Maxisorp, by Thermo Fisher Scientific (4 mentions)
Synergy ht microplate reader, by Agilent Technologies (4 mentions)
Chonblock, by Chondrex (4 mentions)
Hrp goat anti mouse, by BioLegend (3 mentions)
Neutral buffered formalin, by Merck Group (2 mentions)
2 n sulfuric acid, by Thermo Fisher Scientific (2 mentions)
Peroxidase conjugated goat anti mouse igg fab antibody, by Jackson ImmunoResearch (2 mentions)
Prism 8, by GraphPad (1 mentions)
Anti sars cov 2 nucleocapsid, by GeneTex (1 mentions)
Nunc maxisorp, by Thermo Fisher Scientific (1 mentions)
Streptavidin horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Ez link sulfo nhs biotin, by Thermo Fisher Scientific (1 mentions)
Infinite m1000 plate reader, by Tecan (1 mentions)
Stop solution for tmb substrates, by Thermo Fisher Scientific (1 mentions)
Elisa coating buffer, by Bio-Rad (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Monensin, by Merck Group (1 mentions)
Alexa fluor 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti β actin, by Merck Group (1 mentions)
14 protocols using 1 step turbo tmb elisa substrate solution
1
Quantifying Heparan Sulfate Proteoglycan-2
2
ELISA Protocol for Antibody Detection
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ELISAs
were done essentially as previously
described.79 (link) Briefly, plates (Maxisorb)
were coated in 50 μL of 5 μg/mL sfGFP or the RBD for 2
h at room temperature. Plates were washed three times with 1×
PBST and then blocked with 1× PBST with 0.5% bovine serum albumin
(GFP) or ChonBlock (RBD) for at least 1 h at room temperature. Plates
were washed once with 1× PBST, 50 μL of serial dilutions
of guinea pig or mouse serum in 1× PBST was added to the plate
for 1 h at room temperature, and the plates were washed three times
with 1× PBST. An anti-guinea pig horseradish peroxidase secondary
antibody (Abcam) at a 1:10,000 dilution or anti-mouse or anti-mouse
IgG1/IgG2a/IgG2b (Abcam) in 1× PBST was added to each well and
incubated for 1 h at room temperature. Finally, plates were washed
four times with 1× PBST, and 50 μL of 1-Step Turbo TMB-ELISA
Substrate Solution (Thermo Fisher Scientific) was added to each well.
Plates were quenched with 50 μL of 2 M H2SO4 and read on a spectrophotometer. Data were visualized, and EC50 was calculated using GraphPad Prism 8.4.1.
were done essentially as previously
described.79 (link) Briefly, plates (Maxisorb)
were coated in 50 μL of 5 μg/mL sfGFP or the RBD for 2
h at room temperature. Plates were washed three times with 1×
PBST and then blocked with 1× PBST with 0.5% bovine serum albumin
(GFP) or ChonBlock (RBD) for at least 1 h at room temperature. Plates
were washed once with 1× PBST, 50 μL of serial dilutions
of guinea pig or mouse serum in 1× PBST was added to the plate
for 1 h at room temperature, and the plates were washed three times
with 1× PBST. An anti-guinea pig horseradish peroxidase secondary
antibody (Abcam) at a 1:10,000 dilution or anti-mouse or anti-mouse
IgG1/IgG2a/IgG2b (Abcam) in 1× PBST was added to each well and
incubated for 1 h at room temperature. Finally, plates were washed
four times with 1× PBST, and 50 μL of 1-Step Turbo TMB-ELISA
Substrate Solution (Thermo Fisher Scientific) was added to each well.
Plates were quenched with 50 μL of 2 M H2SO4 and read on a spectrophotometer. Data were visualized, and EC50 was calculated using GraphPad Prism 8.4.1.
3
ACE2-NN-Fc Neutralization of SARS-CoV-2
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The neutralization activity of ACE2-NN-Fc variants against SARS-CoV-2 was determined by a microneutralization assay as described elsewhere with modifications [30 (link)]. Briefly, serially diluted proteins were pre-incubated with SARS-CoV-2 (MOI = 0.9) at 37°C for one hour, and the protein-virus mixtures were added to Vero E6-hACE2 cells. The mixtures were removed from cells after one-hour adsorption at 37°C and replaced with virus growth media (1×MEM with 2% FBS and 0.2% BSA) containing the serially diluted proteins. Cells were fixed with 4% formaldehyde at 24 hours post-infection, permeablized with 0.2% Triton X-100, and immunostained with anti-SARS-CoV-2 nucleocapsid (GeneTex 6H3) and HRP-linked anti-mouse IgG (CST #7076). Color was developed by using 1-Step Turbo TMB-ELISA Substrate Solution (Thermo Scientific) and the optical density measured at 652 nm.
4
Phage Display ELISA for Protein Binding
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The binding reactivity of each phage pool to the surface-tethered recombinant ECD was tested in a 96-well ELISA plate (Nunc MaxiSorp, Thermo Fisher Scientific) coated overnight with 10 µg/mL of purified mβc (mu-sAIC2B-His) or mβIL-3 (mu-sAIC2A-His), followed by 3 washes with PBS. Antigen-coated wells and all phage pools, including the original phage scFv library, were blocked with 400 µL 2% MPBS. Next, 200 µL of blocked phage particles were added in duplicate, then serially diluted to 1:1000 across wells and left to incubate for 1 h. Wells were washed 3 times with PBST followed by incubation with a 1:5000 dilution of an anti-M13 mouse antibody for 1 h. Wells were washed 3 times with PBST and incubated with an HRP-conjugated goat anti-mouse IgG secondary antibody at a 1:5000 dilution for 1 h. Wells were washed 3 times with PBST prior to the addition of 100 µL of 1-Step Turbo TMB-ELISA substrate solution (Thermo Fisher Scientific). After 10 min, the reaction was quenched with 100 µL 2 M sulphuric acid, and HRP activity was detected at an absorbance of 450 nm using the PowerWave X52 microplate spectrophotometer (BioTek Instruments Inc., Currumbin, QLD, Australia).
5
SARS-CoV-2 S Protein ELISA
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The 96-well ELISA plates (Sumilon type S, Sumitomobakelight co., ltd, Japan) were coated with 50 µL of 5 µg/mL of the S proteins in bicarbonate buffer (pH9.6) and incubated overnight at 4 °C. The plates were washed with PBS containing 0.1% Tween-20 (PBST) and PBS and blocked with a blocking buffer (PBS containing 1% BSA) at room temperature for 1 h. After washing, 50 µL of each serum sample was added into each well. The plates were washed, and 50 µL of 0.5% BSA containing a peroxidase-conjugated goat anti-mouse IgG Fab antibody (Jackson immunoresearch, Laboratories, Inc., West Grove, PA, USA) was added into each well and incubated for 1 h. Finally, the plates were washed with PBST and PBS, and the color reaction was developed with 1-Step Turbo TMB-ELISA substrate solution (Thermo Scientific). The optical density at 450 nm (OD450nm) was read using a microplate reader.
6
SARS-CoV-2 Protein ELISA Assay
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The 96-well ELISA plates (Sumilon type S, Sumitomobakelight co., ltd, Japan) were coated with 50 or 100 μL of 1 μg/mL of the SARS2/SNFPP+CMP+TEV-H8STREPH6 proteins or SUMO-SARS2/RBD+TEV-H8STREPH6 proteins in bicarbonate buffer (pH9.6) and incubated overnight at 4°C. The plates were washed with PBS containing 0.1% Tween-20 (PBST) and PBS and blocked with a blocking buffer (PBS containing 1% BSA) at room temperature for 1 h. After washing, 50 μL of each diluted serum sample was added to each well. Next, the plates were washed, and 50 μL of 0.5% BSA containing a peroxidase-conjugated goat anti-mouse IgG Fab antibody (Jackson immunoresearch, Laboratories, Inc., West Grove, PA, USA) was added into each well and incubated for 1 h. Finally, the plates were washed with PBST and PBS, and the color reaction was developed with ABTS solution (Figures 5B, D Figures 6B, C
7
Prouroguanylin ELISA Assay
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Mice were fasted for 16 h overnight, blood was collected before and after 1 h of refeeding and serum was isolated. A rabbit anti-prouroguanylin antibody, 6912 (M Goy, University of North Carolina, Chapel Hill, NC, USA11 (link)), was used (1:1000) to coat Nunc-Immuno PolySorp plates (Thermo) for 16 h at 4 °C. The prouroguanylin peptide (CQQKSGLLPDVSYNP) was serially diluted (1:5) in Superblock T20 (phosphate-buffered saline) Blocking Buffer (Thermo) and plated to generate a standard curve (1 μg ml−1 to 2.56 pg ml−1). Serum samples were diluted (1:10) in Superblock and plated. The peptide was biotinylated using EZ-Link Sulfo-NHS-Biotin (Thermo), diluted in Superblock and added to each standard and sample. Plates were sequentially incubated for 30 min at 37 °C, with streptavidin-horseradish peroxidase (Thermo) for 30 min at 37 °C, with 1-Step Turbo TMB-ELISA Substrate Solution (Thermo) for 30 min at room temperature, stopped with 1 M H3PO4 and absorbance quantified at 460 nm.
8
Quantitative Fibronectin Deposition Assay
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Fibronectin deposition was assayed by semiquantitative immunocytochemical detection. Cells were fixed in 10% neutral buffered formalin (Sigma‐Aldrich), endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in methanol for 1 hour, and samples were blocked overnight in 3% goat serum. Samples were then incubated with monoclonal rabbit antifibronectin antibody (polyclonal, 0.4 μg/mL; Santa Cruz Biotechnology) for 2 hours, followed by a series of washes with 1X PBS and labeling with a horseradish peroxidase–linked goat antirabbit secondary antibody (polyclonal, 0.5 μg/mL; Alpha Diagnostic International) for 40 minutes. After washing, samples were incubated in 1‐Step Turbo‐TMB ELISA substrate solution (Thermo Fisher Scientific), and the reaction stopped after 5 minutes through the addition of 2 N sulfuric acid (Thermo Fisher Scientific). Color development was proportional to fibronectin content and measured with a Tecan Infinite M1000 plate reader. Background signal was determined by following the same procedure without incubating in primary antibody solution. This background absorbance was subtracted from the samples for the respective culture condition. Corrected absorbance values were normalized to total cell number determined by DAPI staining and expressed as a fold change over the control condition at each time point.
9
Quantitative Cas9 Antibody Detection
10
Affinity Purification and Antibody Immunodetection
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Anti-FLAG M2 affinity agarose gel, N-ethylmaleimide, sucrose, monensin, Triton X-100, and BSA were purchased from Sigma. The following IgGs were procured from the sources listed: mouse monoclonal c-Myc (catalog no. SC-40) and rabbit polyclonal c-Myc (catalog no. SC-789) from Santa Cruz Biotechnology, Inc.; mouse monoclonal anti-β-actin (catalog no. A5441) from Sigma; anti-ubiquitin FK1 (BML-PW8805) from Enzo Life Sciences; rabbit polyclonal STAMBP (catalog no. 5245), rabbit monoclonal USP14 (catalog no. 11931), rabbit monoclonal USP10 (catalog no. 8501), rabbit monoclonal A20/TNFAIP3 (catalog no. 5630), rabbit polyclonal Myc tag (catalog no. 2272), rabbit monoclonal GAPDH (HRP conjugate, catalog no. 3683), and rabbit polyclonal ubiquitin (catalog no. 3933) antibodies from Cell Signaling Technology; and rabbit polyclonal anti-USP20 (A301-189A) and rabbit polyclonal anti-USP33 (A300-925A) from Bethyl Laboratories, Inc. Dyngo-4a® was purchased from Abcam. 1-StepTM Turbo TMB-ELISA Substrate Solution was purchased from Thermo Scientific. HRP-conjugated secondary antibodies were from GE Biosciences, Cell Signaling Technology and Bethyl Laboratories, Inc. Lipofectamine 2000TM was purchased from Invitrogen. Alexa Fluor 594–conjugated secondary antibody was obtained from Invitrogen and used at a dilution of 1:500 for immunofluorescence labeling.
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