Fifty micrograms of membrane extracts of HEK293T-iUS28 or HEK293T cells transfected with the different US28 ICL mutants were coated overnight in a 96 well MicroWell MaxiSorp flat bottom plate (Sigma-Aldrich, Saint Louis, Missouri, USA). The next day, plates were washed with 1× PBS and blocked with 2% (w/v) skimmed milk in PBS for 1 h at RT. Nanobodies were diluted in 2% (w/v) skimmed milk and incubated for 1 h at RT. Nanobodies were detected with mouse-anti-Myc antibody (1:1000, Clone 9B11, Cell Signaling Technology, Leiden, The Netherlands) and horseradish peroxidase (HRP)-conjugated goat-anti-mouse antibody (1:1000, Bio-Rad). Incubations with antibodies were done for 1 h at RT. Wells were washed three times with 1× PBS between all incubation steps. Binding was determined with 1-step Ultra TMB-ELISA substrate (Thermo Fisher Scientific) and the reaction was stopped with 1 M H2SO4. Optical density was measured at 450 nm with a PowerWave plate reader (BioTek). Data were analyzed using GraphPad Prism version 8.0 (GraphPad Software, Inc., La Jolla, CA, USA).
Microwell maxisorp flat bottom plate
Manufactured by Merck Group
Sourced in United States
MicroWell MaxiSorp flat bottom plates are a type of laboratory equipment designed for various applications. They feature a flat bottom and are made of a material suitable for use in biological and chemical experiments. The core function of these plates is to provide a stable and uniform surface for conducting experiments, though their specific intended use may vary depending on the research or application.
Automatically generated - may contain errors
Lab products found in correlation
Powerwave plate reader, by Agilent Technologies (2 mentions)
Tmb kit, by BD (2 mentions)
1 step ultra tmb elisa substrate, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse antibody, by Bio-Rad (1 mentions)
Clone 9b11, by Cell Signaling Technology (1 mentions)
Goat anti mouse igg hrp conjugate, by Bio-Rad (1 mentions)
Skimmed milk, by Merck Group (1 mentions)
Mouse anti myc antibody, by Cell Signaling Technology (1 mentions)
Np4 bsa, by Biosearch Technologies (1 mentions)
Np26 bsa, by Biosearch Technologies (1 mentions)
Duoset elisa kit, by R&D Systems (1 mentions)
Dy999, by R&D Systems (1 mentions)
5 protocols using microwell maxisorp flat bottom plate
1
Nanobody Binding Assay for US28 Mutants
2
Nanobody Binding Assay for US28 Receptor
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Nanobody binding was performed as described previously14 (link). In brief, US28-expressing membrane extracts were coated in a 96-well MicroWell MaxiSorp flat bottom plate (Sigma-Aldrich) overnight at 4 °C. The next day, wells were washed and blocked with 2% (w/v) skimmed milk (Sigma-Aldrich) in PBS. Different concentrations of nanobodies were incubated. Nanobodies were detected with mouse-anti-Myc antibody (1:1000, clone 9B11, Cell Signaling Technology, Leiden, The Netherlands) and Goat anti-Mouse IgG-HRP conjugate (1:1000, #1706516, Bio-Rad). Optical density was measured at 490 nm with a PowerWave plate reader (BioTek, Winooski, VT, USA). Data were analyzed using GraphPad Prism version 8.0 (GraphPad Software, Inc., La Jolla, CA, USA).
3
Isotype-specific ELISA for Collagen II Abs
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96 well MicroWell MaxiSorp flat bottom plates (Sigma-Aldrich) were coated with 2μg/ml of antigen (CII, Sigma-Aldrich)) and incubated overnight at 4°C. The following day the plates were washed and blocked. Serial serum dilutions were added to the plates and incubated overnight at 4°C. The next day, plates were incubated with anti-mouse IgG1, IgG2a, IgG2b, IgG3 and IgM isotypes coupled to Horse Radish peroxydase (Lo-Imex, Louvain, BE), revealed using a TMB kit (BD Biosciences) and read at 450nm using a ELISA plate reader [15 (link), 16 (link)].
4
Enzyme-Linked Immunosorbent Assay (ELISA) Protocol
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96 well MicroWell MaxiSorp flat bottom plates (Sigma-Aldrich) were coated with 2μg/ml of NP26-BSA, NP4-BSA (Biosearch Technologies, CA, USA) and CGG (Jackson ImmunoResearch) in PBS and incubated overnight at 4°C. The following day the plates were washed with PBS-Tween20 0.1% and blocked with PBS/BSA 1% for 3 hours at room temperature. After removal of blocking solution, serial serum dilutions in PBS/BSA 0.1% were added to the plates and incubated overnight at 4°C. The next day, plates were washed with PBS-Tween20 0.1% and incubated with anti-mouse IgG1, IgG3 and IgM isotypes coupled to Horse Radish peroxydase (Lo-Imex, Louvain, BE) for 2 hours at 37°C. After this incubation, plates were washed and revealed using a TMB kit (BD Biosciences). The reaction was stopped using a 2M sulfuric acid solution and the plates were read at 450nm.
5
Quantification of Astrocyte Cytokines
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Cytokine and chemokine release from astrocytes were quantified using commercially available duo-set ELISA kits (R&D Systems) for mouse IL-6 (DY406), TNF-α (DY410), IL-1β/ IL-1F2 (DY401) and CX3CL1 (DY472). Briefly, 96 well MicroWell™ MaxiSorp™ flat bottom plates (Sigma, 442404) were coated with the capture antibody diluted in PBS for 18 h at 4°C. Plates were then washed in 0.5% Tween-20 in PBS and blocked with the appropriate reagent diluent for 1 h at 22°C. The blocking buffer was then removed and the astrocyte culture medium and recombinant protein standards were diluted, added to the wells and incubated for 2 h at 22°C. The wells were then washed and the detection antibody was added for 2 h at 22°C. The detection antibody was then washed off and a solution containing streptavidin-HRP secondary antibody was added for 20 min and kept in the dark. The secondary antibody solution was washed off and the substrate solution was added (DY999, R&D Systems) and incubated for 20 min. The reaction was stopped by adding 2N H2SO4 to the plate and absorbance was immediately read at 450 nm with wavelength correction at 570 nm.
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