Antigen-specific Immunoglobulin G (IgG) titers were determined using MSD electrochemical luminescence (MSD-ECL) assays according to the manufacturer's instructions. The MSD-ECL plates were printed with FHA, FIM2/3, PRN, PTxd, TT, DT antigens (multi-spot® 96-well-7 spot plates) by the manufacturer (Meso Scale Discovery). Serum IgG was detected using goat anti-pig IgG (Fc) biotin conjugate (Biorad #AAI41B) followed by Streptavidin-Sulfo-Tag® (MSD #R32AD-1).
Streptavidin sulfo tag
Manufactured by MSD
Streptavidin-Sulfo-Tag is a reagent that contains streptavidin conjugated to a Sulfo-TAG label. Streptavidin is a tetrameric protein that binds strongly to the vitamin biotin. The Sulfo-TAG label is a detection system that can be used in various biochemical and immunological assays.
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5 protocols using streptavidin sulfo tag
1
Quantification of Antigen-Specific IgG Titers
2
Multiplexed Antibody Isotyping and IgE Quantification
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IgA, IgG1, IgG2a, IgG2b, IgG3 and IgM were measured using a kit (Mouse Isotyping Panel 1 kit) from MSD according to their protocol. Plasma samples were diluted 1∶1000, to 1∶125,000 to ensure samples reading within the linear range of the assay for each antibody.
To measure total IgE, standard 96-well MSD plates were coated (overnight, 4°C) with anti-IgE (2 µg/ml, PBS, BD). All further incubations were carried out at room temperature on an orbital shaker. Unbound antibody was removed by washing (0.05% Tween 20 in PBS), and non-specific binding blocked (1% BSA/PBS, 200 µl/well) for 1 h. The plate was washed, 25 µl standard (14–10,000 ng/ml, purified mouse IgE, BD) or sample (diluted 1∶10) added and incubated for 2 h. Unbound antibody was removed by washing and 25 µl biotinylated anti-mouse IgE (1 µg/ml; BD) pipetted per well. Following 1 h incubation, the plates were washed and 25 µl streptavidin Sulfo-Tag (1 µg/ml; MSD) added for a further 1 h. Plates were then washed, 150 µl Read buffer (T- x2; MSD) added per well and the samples analysed on SI6000 (MSD). OVA-specific IgE was measured by ELISA (AbD Serotec) according to their protocol.
To measure total IgE, standard 96-well MSD plates were coated (overnight, 4°C) with anti-IgE (2 µg/ml, PBS, BD). All further incubations were carried out at room temperature on an orbital shaker. Unbound antibody was removed by washing (0.05% Tween 20 in PBS), and non-specific binding blocked (1% BSA/PBS, 200 µl/well) for 1 h. The plate was washed, 25 µl standard (14–10,000 ng/ml, purified mouse IgE, BD) or sample (diluted 1∶10) added and incubated for 2 h. Unbound antibody was removed by washing and 25 µl biotinylated anti-mouse IgE (1 µg/ml; BD) pipetted per well. Following 1 h incubation, the plates were washed and 25 µl streptavidin Sulfo-Tag (1 µg/ml; MSD) added for a further 1 h. Plates were then washed, 150 µl Read buffer (T- x2; MSD) added per well and the samples analysed on SI6000 (MSD). OVA-specific IgE was measured by ELISA (AbD Serotec) according to their protocol.
3
Quantification of Tau Phosphorylation Levels
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HEK cell lysates and human brain extracts were diluted with 1% bovine serum albumin (BSA) in wash buffer (TBS supplemented with 0.03% Tween). For pT175, pT181, pT217, pT231, N‐terminal tau, AT8, and pTPP assays, each well of an uncoated 96‐well multi‐array plate (MesoScale Discovery) was coated with 30 μL of a PBS solution containing 1 μg/mL of Tau‐13 capture antibody to the N‐terminus region of Tau (epitope aa 15–25) and incubated at room temperature (RT) overnight. The aforementioned detection antibody solutions were prepared to have biotinylated monoclonal antibodies explicitly recognizing the phospho‐epitopes or general epitope (for N‐terminal tau assay), plus 100 ng/mL Streptavidin Sulfo‐TAG (MSD, No. R32AD‐5) and 1% BSA diluted in wash buffer. Following overnight incubation at RT, 50 μL/well of the sample, followed by 25 μL/well of detection antibody solution were incubated for 2 h at RT with shaking at 850 rpm and washing of wells with wash buffer between incubations. The plate was read and analyzed according to the MSD manufacturer's protocol. Full‐length tau was phosphorylated by GSK‐3β through their co‐expression in BL21 (DE3) Escherichia coli cells and purified for use as a calibrator for all assays.
4
Quantitative BTK Phosphorylation Assay
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Human or mouse whole blood was treated with titrating concentrations of BIIB091 or dimethyl sulfoxide (DMSO) (Sigma‐Aldrich, St. Louis, MO) for 30 min and subsequently lysed at room temperature (RT) for 10 min using lysis buffer (150 mm NaCl, 20 mm Tris pH 7.5, 1 mm EDTA, 1 mm EGTA, 1% Triton‐X‐100) containing protease and phosphatase inhibitors (MSD, Rockville, MD). Negative control (DMSO‐treated) samples were lysed in buffer containing protease inhibitors but lacking phosphatase inhibitors. Lysates were diluted (1:2) in Blocking Buffer (3% Blocker A; MSD) and transferred to plates (goat anti‐rabbit, MSD) which had been coated overnight (4°C) with 0.5 μg mL−1 of rabbit anti‐BTK (D3H5; Cell Signaling Technologies, Danvers, MA) and subsequently blocked with blocking buffer for 2–3 h at RT. Following overnight (4°C) sample incubation, the plate was incubated for 2 h (RT) with biotinylated anti‐phosphotyrosine (diluted 1:400, P‐Tyr‐100; Cell Signaling Technologies) and 1 h (RT) with streptavidin Sulfo‐Tag (diluted 1:500; MSD). Phosphorylated BTK was detected as ECL signal on a QuickPlex Imager (MSD) after the addition of 1× MSD Read Buffer.
5
Quantifying Biotinylated Transferrin in Cells
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The content of biotinylated Tf in cell lysates was measured in an electrochemiluminescence-based assay using Meso Scale Discovery (MSD) technology platform. The extracts were applied to avidine-coated standard plates (MSD) for 1 h on a shaker platform, followed by addition of streptavidin SULFO-TAG (R32AD-1; MSD) for 1 h and three washes with MSD Tris wash buffer (R61TX-1). Afterwards, the MSD Read Buffer (R92TC-2) was added and electrochemiluminescence of the samples was read in the SECTOR Imager 2400 (MSD).
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