One step ultra tmb substrate

Manufactured by Thermo Fisher Scientific

One-step Ultra TMB substrate is a ready-to-use substrate solution for the detection of horseradish peroxidase (HRP) in immunoassays. It provides a colorimetric signal in the presence of HRP.

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2030 multilabel reader victor x5, by PerkinElmer (1 mentions)

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TMB substrate (351 citations) Ultra-TMB substrate (33 citations) Ultra-TMB (22 citations) One-Step Ultra TMB (10 citations) One-Step Ultra TMB ELISA HRP substrate solution (6 citations) One-Step TMB (4 citations) One-step TMB substrate (3 citations) One-step™ Ultra TMB-ELISA Substrate Solution (3 citations)

6 protocols using one step ultra tmb substrate

Quantification of Plasma and Vaginal SHIVIG

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The concentration of SHIVIG in plasma samples was determined by ELISA. The 96-well plates were coated with gp120 of HIV-1_BaL at 0.6 μg/ml for plasma SHIVIG determination and 1 μg/ml for measurement of SHIVIG content in vaginal lavages. Plates were incubated overnight at 4°C. After blocking and washing, serially diluted, heat-inactivated plasma samples were added to plates in triplicates. Vaginal lavage samples were thawed, diluted 1:1 with blocking buffer, heat-inactivated and added to plates in triplicate. SHIVIG was included as a standard ranging from 0.156 to 1 μg/ml. To detect binding, plates were incubated with rabbit anti-monkey IgG HRP-conjugated Ab and developed with One Step Ultra TMB Substrate (Thermo Fisher Scientific). To determine the half-life of SHIVIG, natural logs of SHIVIG plasma levels were plotted as a function of time from the end of infusion. Slopes of the linear graphs were determined by least-squares analysis. Half-lives were calculated as t_1/2 = −(ln 2)/m.
Total IgG content in vaginal lavage fluids was assessed by ELISA. Briefly, plates were coated with rabbit anti-monkey IgG-whole molecule (Sigma), blocked and probed with serially diluted vaginal lavage samples in triplicates. After washing, bound IgG was detected with rabbit anti-monkey IgG HRP-conjugated Ab and developed with One Step Ultra TMB Substrate. Naïve RM (nRM) IgG served as a standard.

Sholukh A.M., Byrareddy S.N., Shanmuganathan V., Hemashettar G., Lakhashe S.K., Rasmussen R.A., Watkins J.D., Vyas H.K., Thorat S., Brandstoetter T., Mukhtar M.M., Yoon J.K., Novembre F.J., Villinger F., Landucci G., Forthal D.N., Ratcliffe S., Tuero I., Robert-Guroff M., Polonis V.R., Bilska M., Montefiori D.C., Johnson W.E., Ertl H.C, & Ruprecht R.M. (2014). Passive immunization of macaques with polyclonal anti-SHIV IgG against a heterologous tier 2 SHIV: outcome depends on IgG dose. Retrovirology, 11, 8.

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ELISA for Detecting Antibodies Against hVLPs

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ELISAs on directly immobilized hVLPs were performed as previously described (Tong et al., 2012 (link)). Briefly ADA.CM.v4 hVLPs (prepared as detailed above and treated with or without AT-2) were immobilized on microwell plates at a 20x concentration for 2 h at 37þC. Plates were blocked with 4% non-fat dry milk (NFDM) PBS for 1 h at 37þC, probed with serial dilutions of primary antibodies and, subsequently, goat anti-human-Fcɣ-HRP secondary antibody (Jackson) in PBS + 0.4% NFDM, and with washes using PBS between each step (detergent was omitted from all steps). Signal was developed using One-step Ultra TMB Substrate (ThermoFisher).

Mangala Prasad V., Leaman D.P., Lovendahl K.N., Croft J.T., Benhaim M.A., Hodge E.A., Zwick M.B, & Lee K.K. (2022). Cryo-ET of Env on intact HIV virions reveals structural variation and positioning on the Gag lattice. Cell, 185(4), 641-653.e17.

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Phospho-Stat5a/b Quantification Assay

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T47D cells in 96-well plates were serum-starved for 16 h, pretreated with IST-002 at the indicated concentrations for 2 h, followed by stimulation with hPrl (2 nM) for 15 min, and fixation in 20°C acetone/methanol followed by incubation with anti-pStat5a/b rabbit mAb (1:200; Cell Signaling) and secondary HRP-conjugated anti-rabbit Ab (1:2000, Cell Signaling). The amount of phospho-Stat5a/b was evaluated using one-step Ultra TMB substrate (Thermo Scientific) at 450 nm. Cells cultured in the absence of IST-002 and treated with or without prolactin served as positive and negative controls, respectively. The inhibition curve and IC50 were derived from triplicate measurements using Sigmaplot.

Liao Z., Gu L., Vergalli J., Mariani S.A., De Dominici M., Lokareddy R.K., Dagvadorj A., Purushottamachar P., McCue P.A., Trabulsi E., Lallas C.D., Gupta S., Ellsworth E., Blackmon S., Ertel A., Fortina P., Leiby B., Xia G., Rui H., Hoang D.T., Gomella L.G., Cingolani G., Njar V., Pattabiraman N., Calabretta B, & Nevalainen M.T. (2015). Structure-based screen identifies a potent small-molecule inhibitor of Stat5a/b with therapeutic potential for prostate cancer and chronic myeloid leukemia. Molecular cancer therapeutics, 14(8), 1777-1793.

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ELISA for Anti-dsDNA Antibody Quantification

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Mouse IgG anti-dsDNA antibodies were measured by ELISA as previously described [11 (link)]. Briefly, Costar (Corning, NY) 96 well flat bottom microtiter plates were coated overnight at 4°C with 10 μg/ml dsDNA in PBS, pH 7.2. Various dilutions of mouse sera or murine monoclonal IgG anti-dsDNA antibody ( Millipore, Billerica, MA) standard were added in PBS and incubated overnight at 4° C. Bound anti-dsDNA antibodies were detected using HRP-goat anti-mouse IgG-Fc-specific (Bethyl Labs, Montgomery, TX) antibodies and One Step Ultra TMB substrate (Thermo, Rockford, IL) and measured at 450nm [11 (link)].

Strickland F.M., Li Y., Johnson K., Sun Z, & Richardson B.C. (2015). CD4+ T Cells Epigenetically Modified by Oxidative Stress Cause Lupus-like Autoimmunity in Mice. Journal of autoimmunity, 62, 75-80.

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SARS-CoV-2 Antibody Detection by ELISA

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Patient plasma samples were tested for the presence of anti-SARS-CoV-2 reactive IgM or IgG antibodies as described previously (Snyman et al., 2021 (link)). ELISA plates were coated with 500 ng/ml of the D614G ancestral virus receptor binding domain (RBD) (GenBank: MN975262; provided by Dr Galit Alter, Ragon Institute, Cambridge, MA, USA) overnight at 4°C. Then blocked with 1% BSA-TBS at room temperature (RT) for 1 hr, followed by samples diluted at 1:100 in BSA-TBS+0.05% Tween 20 for 1 hr at RT. Secondary anti-IgM or -IgG antibodies (Jackson ImmunoReasearch, West Grove, PA, USA) were added at 1:5000 diluted in BSA-TBS+0.05% Tween 20 and incubated again for 1 hr at RT. Finally, plates were developed with one-step Ultra TMB substrate (Thermo Fisher Scientific) for 3 or 5 min respectively and signal development was stopped with the addition of 1 N H₂SO₄. Plates were washed with TBS+0.05% Tween 20 between each incubation step. All signals were compared to anti-SARS-CoV-2-specific monoclonal IgG (clone CR3022) or IgM (clone hIgM2001). Pre-pandemic plasma samples were used as negative controls to determine seroconversion cut-offs calculated as three times the standard deviation plus the mean.

Krause R., Snyman J., Shi-Hsia H., Muema D., Karim F., Ganga Y., Ngoepe A., Zungu Y., Gazy I., Bernstein M., Khan K., Mazibuko M., Mthabela N., Ramjit D., Limbo O., Jardine J., Sok D., Wilson I.A., Hanekom W., Sigal A., Kløverpris H., Ndung'u T, & Leslie A. (2022). HIV skews the SARS-CoV-2 B cell response towards an extrafollicular maturation pathway. eLife, 11, e79924.

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GRP78 Binding Assay using OxPAPS and DMPS

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Microtiter 96-well plates (MaxiSorp; Nunc, Thermo Scientific, Rochester, NY) were coated with OxPAPS or DMPS (each 100 μg/ml in PBS containing 0.01% BHT) at 4°C overnight. Plates were washed with PBS and blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Recombinant human GRP78 (Prospec-Tany Technogene, East Brunswick, NJ; 1 μg/ml in PBS/1% BSA) or HPAEC cell lysates in PBS/0.05% Tween-20 were applied for 1 h at room temperature. After washing with PBS and sequential addition of anti-GRP78 and anti-goat horseradish peroxidase (HRP) antibodies, one-step Ultra-TMB substrate (Thermo Scientific) was added. Absorption was measured using a 2030 Multilabel Reader Victor X5 (PerkinElmer, Waltham, MA).

Birukova A.A., Singleton P.A., Gawlak G., Tian X., Mirzapoiazova T., Mambetsariev B., Dubrovskyi O., Oskolkova O.V., Bochkov V.N, & Birukov K.G. (2014). GRP78 is a novel receptor initiating a vascular barrier protective response to oxidized phospholipids. Molecular Biology of the Cell, 25(13), 2006-2016.

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