Substrate reagent pack

Manufactured by R&D Systems

Sourced in United States, United Kingdom

The Substrate Reagent Pack is a laboratory product that provides a substrate solution for use in various biochemical and immunological assays. The pack contains the necessary reagents to prepare the substrate solution, enabling researchers to conduct their experiments efficiently.

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Lab products found in correlation

19 protocols using substrate reagent pack

Selectin-Sialyl Lewisx Binding Assay

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Recombinant human L-selectin (10 ng/ml), P-selectin (10 ng/ml) or E-selectin (30 ng/ml) Fc chimeras (R&D Systems) were mixed with increasing concentrations of heparin or heparin derivatives in PBS before addition to 96-well ELISA plates precoated with BSA-Sialyl Lewis^x (110 ng/well, R&D Systems) for 45 minutes at room temperature. The plates were washed three times with 200 μl of Quantikine wash buffer (R&D Systems) and the bound selectins were detected using an HRP-anti Fc antibody and Substrate Reagent Pack (R&D Systems) according to the manufacturer's instructions.

Duckworth C.A., Guimond S.E., Sindrewicz P., Hughes A.J., French N.S., Lian L.Y., Yates E.A., Pritchard D.M., Rhodes J.M., Turnbull J.E, & Yu L.G. (2015). Chemically modified, non-anticoagulant heparin derivatives are potent galectin-3 binding inhibitors and inhibit circulating galectin-3-promoted metastasis. Oncotarget, 6(27), 23671-23687.

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Quantifying Anti-Rapamycin Antibodies

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Plates were coated with rapamycin or rapamycin nanoparticles overnight. The next day plates were washed three times with 0.05% tween-20 in PBS, blocked with 1% BSA/PBS for 1 h, and washed again three times. Serum samples were diluted 1:100 in 1% BSA/PBS; 100 μL was added to each well and incubated for 2 h at room temperature. After washing three times, 100 μL of either IgG or IgM HRP conjugated antibody (goat anti-mouse IgG/IgM-HRP, Cat #’s: 1030-05, 1020-05. Southern Biotechnology Associates. Birmingham, AL, USA) was added to each well at a 1:3000 dilution and incubated for 2 h at room temperature. The plates were washed and developed according to the protocol of the substrate manufacturer (Substrate Reagent Pack, Cat No. DY999, R&D Systems, Minneapolis, MN, USA) and read at 450 nm absorbance using a Spectra Max Plus.

Zhou Q., Doherty J., Akk A., Springer L.E., Fan P., Spasojevic I., Halade G.V., Yang H., Pham C.T., Wickline S.A, & Pan H. (2022). Safety Profile of Rapamycin Perfluorocarbon Nanoparticles for Preventing Cisplatin-Induced Kidney Injury. Nanomaterials, 12(3), 336.

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Multiplex Analysis of Serum Immune Factors

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Chemokines and other soluble factors from serum were assessed in multiplex immunoassays utilizing Luminex technology. Serum from ILP patients and from healthy donors were analyzed with a 14-plex immune checkpoint panel (Human Immuno-Oncology Checkpoint Marker Panel, Invitrogen, #EPX14A-15803-901) and a 27-plex cytokine panel (Bio-Plex™ Pro Human Cytokine Standard 27-Plex Group 1, Bio-Rad, #M500KCAF0Y) according to the manufacturers’ protocols. All measurements were performed using a Bio-Plex™ 200 System (Bio-Rad).
Soluble CXCL10, CCL2, IFN-γ, IFN-α2 and IFN-β in cell culture supernatants were measured using a CXCL10 ELISA kit (Human CXCL10/IP-10 DuoSet ELISA, R&D Systems, #DY266-05), a CCL2 ELISA kit (Human CCL2/MCP-1 DuoSet ELISA, R&D Systems, #DY279-05) and ELISA kits for IFN-γ, IFN-α2 and IFN-β (Human IFN-gamma/IFN-alpha 2/IFN-beta DuoSet ELISA, R&D Systems, #DY285B-05/#DY9345-05/#DY814-05) together with Substrate Reagent Pack (R&D Systems, #DY999) in 96-well plates (Corning™ 96-Well Half-Area Plates, Fisher Scientific, #10052511) according to manufacturer’s protocol. For samples with protein levels lower than the detection limit, 50% of the lowest detected concentration was utilized in statistical calculations.

Johansson J., Kiffin R., Aydin E., Nilsson M.S., Hellstrand K., Lindnér P., Naredi P., Olofsson Bagge R, & Martner A. (2019). Isolated limb perfusion with melphalan activates interferon-stimulated genes to induce tumor regression in patients with melanoma in-transit metastasis. Oncoimmunology, 9(1), 1684126.

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Quantitative Analysis of Growth Factors in 3D Hydrogel Cultures

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Medium was collected from hydrogel cell constructs on days 3, 6, 9, and 12 of culture and stored at − 80 °C until analysis. Total TGF-β1 levels were assessed using Human TGF-beta 1 DuoSet ELISA (DY240; R&D Systems, Minneapolis, MN). Similarly, GDF-15 expression was analyzed using Human GDF-15 DuoSet ELISA (DY957; R&D Systems). For the activation of latent TGF-β1, the medium was treated with Sample Activation Kit (DY010; R&D Systems). TGF-β1 and GDF-15 ELISA were performed using Substrate Reagent Pack (DY999; R&D Systems), and absorbance measurements were made at 450 nm with a 570 nm correction using a SpectraMax i3x Multi-Mode Microplate Reader. α-Amylase expression was quantified using the Human Salivary Amylase Alpha ELISA Kit (NBP2-68203; Novus Biologicals, Littleton, CO), and luminesce measurements were made using a SpectraMax i3x Multi-Mode Microplate Reader. All assays were performed in accordance with the manufacturers’ protocols. Protein levels were normalized to the number of cells in each hydrogel at each time point using the procedure detailed in the 3D Proliferation method conducted on days 1, 3, 6, 9, and 12 of culture.

Fowler E.W., van Venrooy E.J., Witt R.L, & Jia X. (2022). A TGFβR inhibitor represses keratin-7 expression in 3D cultures of human salivary gland progenitor cells. Scientific Reports, 12, 15008.

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Quantitative ELISA for LCMV Antibodies

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96-well polystyrene ELISA plates (Nunc) were coated with LCMV clone 13-infected BHK-21 cell lysate overnight at room temperature. After blocking with PBS supplemented with 10% FCS, 0.2% Tween 20, and 0.5 mM thimerosal, three-fold serial dilutions of serum samples were incubated for 90 min at room temperature. Plates were washed with PBS supplemented with 0.5% Tween 20 and incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse IgGγ, IgG1, IgG2b, IgG2c, and IgG3 antibodies (SouthernBiotech). After washing, plates were incubated with stabilized hydrogen peroxide and tetramethylbenzidine (Substrate Reagent Pack; R&D systems) according to the manufacturer’s instructions, and the reaction was stopped by adding 2N H₂SO₄. Optical density (OD) values at 450 nm were determined using a microplate reader (Molecular Devices).

Tian Y., Mollo S.B., Harrington L.E, & Zajac A.J. (2016). IL-10 regulates memory T cell development and the balance between Th1 and Tfh responses during an acute viral infection. Journal of immunology (Baltimore, Md. : 1950), 197(4), 1308-1321.

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TGF-β and Gremlin Quantification

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The levels of total TGF-β were measured using a TGF-β1 duo-set (DY240) as previously described [11 (link)] while gremlin levels were measured with the gremlin duo-set (DY956) and a substrate reagent pack (DY999) according to the manufacturer’s instructions (R&D Systems Europe, Abingdon, UK).

Ouahoud S., Westendorp B.F., Voorneveld P.W., Abudukelimu S., Koelink P.J., Pascual Garcia E., Buuren J.F., Harryvan T.J., Lenos K.J., van Wezel T., Offerhaus J.A., Fariña-Sarasqueta A., Crobach S., Slingerland M., Hardwick J.C, & Hawinkels L.J. (2022). Loss of bone morphogenetic protein signaling in fibroblasts results in CXCL12-driven serrated polyp development. Journal of Gastroenterology, 58(1), 25-43.

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Quantifying PD-1/PD-L1 Binding Interactions

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Streptavidin-HRP (cat# DY998), substrate reagent pack (cat# DY999), and stop solution (cat# DY994) were purchased from R&D Systems (Minneapolis, MN). Ninety-six-well plates were coated with 100 ng of human PD-L1 ECD (cat# FCL0784, G&P Biosciences) or mouse PD-L1 ECD (cat# FCL3502, G&P Biosciences) overnight at 4°C and blocked with 2% BSA for 2 h at room temperature. dAbs were loaded in each well and incubated for 1 h at room temperature. Next, unbound dAbs were removed by washing with PBS. Five hundred nanograms of biotinylated human PD-1 ECD (cat# FCL0761B, G&P Biosciences) or mouse PD-1 (cat# FCL1846B, G&P Biosciences) was loaded in each well and incubated for 1 h. After washing with PBS, 100 µl of Streptavidin-HRP (diluted 1:200 in 1% BSA) was added and incubated for 20 min, followed by the addition of substrate reagents and stop solution. The absorbance at 450 nm was recorded with the reference wavelength at 540 nm.

Liu H., Liu Y., Zhao Z., Li Y., Mustafa B., Chen Z., Barve A., Jain A., Yao X., Li G, & Cheng K. (2022). Discovery of Anti-PD-L1 Human Domain Antibodies for Cancer Immunotherapy. Frontiers in Immunology, 13, 838966.

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Quantifying Mucosal IgA Antibodies

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Ninety-six-well plates (Thermo Fisher Scientific) were coated with purified anti-IgA (RMA-1, BD) or 0.5nM ml GM1 followed by 0.5 ug/ml CT overnight at 4 °C (47 (link)). The plates were washed and clocked with PBS/5% BSA before diluted fecal samples were added and twofold serial dilution was made. Samples were incubated overnight at 4 °C, followed by biotinylated anti-mouse antibodies: anti-IgA (C10-1, BD), anti-IgA^a and anti-IgA^b (Hy16 and HISM2, UCSF Hybridoma Core) at 1 ug/ml in PBS/0.1% BSA. Detection antibodies were labeled by streptavidin-conjugated horseradish peroxidase (HRP), and visualized by the addition of Substrate Reagent Pack (R&D). Color development was stopped with 3 M H₂SO₄. Purified mouse IgA (Southern Biotech) served as standard and was purchased from Southern Biotech. Absorbances at 450 nm were measured on a tunable microplate reader (VersaMax, Molecular Devices). Antibody titers were calculated by extrapolating absorbance values from standard curves where known concentrations were plotted against absorbance using SoftMax Pro 5 software.

Reboldi A., Arnon T.I., Rodda L.B., Atakilit A., Sheppard D, & Cyster J.G. (2016). IgA production requires B cell interaction with subepithelial dendritic cells in Peyer’s patches. Science (New York, N.Y.), 352(6287), aaf4822.

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Quantifying Fecal Albumin and Lipocalin-2

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Fecal pellets were collected, weighed and resuspended in PBS containing 1% FBS. A sandwich ELISA for fecal albumin and lipocalin-2 was performed on 96-flat bottom Nunc MaxiSorp plates (eBioscience). A purified anti-mouse albumin capture antibody (Bethyl Laboratories, Montgomery, TX) and a secondary HRP-conjugated anti-mouse albumin antibody (Bethyl Laboratories) were used for the capture and detection of fecal albumin. A Substrate Reagent Pack (R&D Systems, Minneapolis, MN) was used for the color reaction and 1 M sulfuric acid was added to stop color development. Colorimetric signals were measured on a SpectraMax M2e device (Bucher Biotec AG, Basel, Switzerland). Fecal lipocalin-2 was measured using the mouse Lipocalin-2/NGAL MAb ELISA kit (R&D systems), according to the manufacturer’s instructions.

Mager L.F., Koelzer V.H., Stuber R., Thoo L., Keller I., Koeck I., Langenegger M., Simillion C., Pfister S.P., Faderl M., Genitsch V., Tcymbarevich I., Juillerat P., Li X., Xia Y., Karamitopoulou E., Lyck R., Zlobec I., Hapfelmeier S., Bruggmann R., McCoy K.D., Macpherson A.J., Müller C., Beutler B, & Krebs P. (2017). The ESRP1-GPR137 axis contributes to intestinal pathogenesis. eLife, 6, e28366.

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Quantifying Cellular HSP27 Secretion

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HSP27 concentration in cell culture supernatant was determined by use of the DuoSet ELISA kit human HSP27 (DY1580; R&D, Minneapolis, MN, USA) with Substrate Reagent Pack containing stabilized hydrogen peroxide (Part #895000; R&D) and stabilized tetramethylbenzidine (Part #895001; R&D) according to the manufacturer's instructions. Duplicates of each sample were measured. Cells and cellular debris were sedimented (1, 3 ×g, 5 min), the supernatant was incubated with capture antibodies (overnight, 4°C), and soluble HSP27 was determined using a BMG FLUOstar OPTIMA Microplate Reader with OPTIMA software 2.10 (BMG Labtech, Offenbach, Germany).

Stope M.B., Klinkmann G., Diesing K., Koensgen D., Burchardt M, & Mustea A. (2017). Heat Shock Protein HSP27 Secretion by Ovarian Cancer Cells Is Linked to Intracellular Expression Levels, Occurs Independently of the Endoplasmic Reticulum Pathway and HSP27's Phosphorylation Status, and Is Mediated by Exosome Liberation. Disease Markers, 2017, 1575374.

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