Phosphatase substrate solution

Manufactured by Merck Group

Sourced in United States

Phosphatase substrate solution is a laboratory reagent used to detect the presence of phosphatase enzymes in biological samples. It provides a chromogenic or fluorogenic substrate that is cleaved by the phosphatase, producing a detectable signal. The solution is designed for use in various biochemical and cell-based assays.

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3 protocols using phosphatase substrate solution

Serum Immunoglobulin and Polyreactivity Analysis

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Total serum Ig was measured by sandwich ELISA. High-binding assay plates were coated overnight with 50 µl capture antibody (Bethyl Labs) diluted in carbonate buffer. Plates were washed with wash buffer (0.1% Tween20 in PBS) and blocked for 2 h at 37°C with 50 µl of 5% BSA and 0.05% sodium azide in PBS. After washing, 50-µl mouse serum samples (diluted in 1% BSA and 0.1% Tween20 in PBS) were added and incubated for 2 h at 37°C. Plates were washed and incubated with alkaline phosphatase–conjugated detection antibodies (Bethyl Labs) for 1 h. Plates were washed and incubated with 100 µl phosphatase substrate solution (Sigma) for 10–15 min, and absorbance was measured at 405 nm.
Polyreactivity was assessed by performing ELISA as previously described (Gitlin et al., 2016 (link)) using cardiolipin (Sigma), LPS (Sigma), human insulin (Sigma), double-stranded DNA (Sigma), and KLH (Sigma).
SRBC-specific IgM, IgG3, and IgG1 in the sera were measured by flow cytometry–based MFIs (median fluorescence intensity) of anti-IgM (Clone II/41; eBioscience), anti-IgG3 (Clone R40-82; BD PharMingen), and anti-IgG1 (A85-1; BD Biosciences) binding to SRBC-bound serum antibodies using a method described previously (McAllister et al., 2017 (link)).

Bhat N., Virgen-Slane R., Ramezani-Rad P., Leung C.R., Chen C., Balsells D., Shukla A., Kao E., Apgar J.R., Fu M., Ware C.F, & Rickert R.C. (2021). Regnase-1 is essential for B cell homeostasis to prevent immunopathology. The Journal of Experimental Medicine, 218(5), e20200971.

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Colorimetric Alkaline Phosphatase Assay

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To quantify the ALP activity in the samples, we used a colorimetric method based on the conversion of p-nitrophenyl phosphate to p-nitrophenol in the presence of ALP. We lysed samples cultured with the extracts and with osteogenic medium with 200 μl of 0.1% Triton X-100 in 1xTE buffer (Sigma-Aldrich, Sant Louis, USA), followed by three freeze–thaw cycles. Subsequently, 50 μl of sample were combined with 50 μl of a mixture in 1:1:1 ratios of 1.5 M 2-amino-2-methyl-1-propanol buffer (Sigma-Aldrich, Sant Louis, USA), 20 mM phosphatase substrate solution (Sigma-Aldrich, Sant Louis, USA) and 1 mM MgCl₂. The samples were incubated for 30 min at 37 °C, and we stopped the reaction using 1 M NaOH. The production of p-nitrophenol was quantified by measuring the absorbance at 405 nm and comparing it against a standard curve prepared with known concentrations of p-nitrophenol. Results were then normalized to the results obtained in the proliferation assay. Cell proliferation was quantified by measuring the amount of dsDNA in the samples with the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Waltham, USA) following the recommendations of the manufacturer. We mixed 100 μl of sample and 100 μl of a 1:200 dilution of PicoGreen reagent in a black 96-well plate.

Toledano-Serrabona J., Bosch B.M., Díez-Tercero L., Gil F.J., Camps-Font O., Valmaseda-Castellón E., Gay-Escoda C, & Sánchez-Garcés M.Á. (2022). Evaluation of the inflammatory and osteogenic response induced by titanium particles released during implantoplasty of dental implants. Scientific Reports, 12, 15790.

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Quantitative ALP Induction by rhBMP-2

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The quantitative analysis of rhBMP-2 on the induction of alkaline phosphatase (ALP) activity in hFOB 1.19 was determined by the modified colorimetric method (Wanachewin et al., 2015) (link). The cells were seeded as previously described and replaced with rhBMP-2 or E. coli-derived hBMP-2 (R&D Systems, USA) in a medium containing 2% FBS. After treatment for 5 days, the cells were washed and the attached cells were scraped off. The cells were lysed with 100 l phosphatase buffer followed by sonication and the protein concentration was determined using Bradford assay. ALP activity assay was performed in a 96-well plate by adding the cell lysate with 150mM alkaline buffer solution (Sigma-Aldrich, USA). The reaction was initiated by adding 1 mg/ml phosphatase substrate solution (Sigma-Aldrich, USA) and incubated at 37C for 60 min. The reaction was stopped by 0.1N NaOH when the color intensity was measured at 405 nm using a microplate reader. The alkaline phosphatase activity was quantified by the production of p-nitrophenol (M) product per mg protein per minute and compared as a percentage with the untreated control (100%).

Kasekarn W., Suksiriphattanapong B., Chokepaichitkool T., Wanachewin O., Roytrakul S., & Kongtawelert P. (2020). Soluble Expression and Purification of Bioactive Recombinant Human Bone Morphogenetic Protein-2 from Escherichia coli. Chiang Mai University Journal of Natural Sciences, 19(4).

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