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Sigmafast p nitrophenyl phosphate tablets

Manufactured by Merck Group
Sourced in United States

SIGMAFAST™ p-Nitrophenyl phosphate Tablets are a colorimetric substrate that can be used to detect and quantify the presence of alkaline phosphatase (ALP) in various assays and applications. The tablets contain p-nitrophenyl phosphate, which is hydrolyzed by ALP to produce a yellow-colored p-nitrophenol product that can be measured spectrophotometrically.

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38 protocols using sigmafast p nitrophenyl phosphate tablets

1

Enzymatic Assay for Alkaline Phosphatase Activity

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ALP activity was measured using SIGMAFAST™ p-Nitrophenyl phosphate Tablets (catalog no. N1891, Merck). Substrate was prepared according to manufacturer’s instructions and mixed with samples. Incubation was performed at 37C for 30 min. Absorbance was measured at 405 nm. Activity was calculated using the following formula
(ΔAbs/min) x Volume x 1,00018.8 x light passage x volume
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2

Quantitative Alkaline Phosphatase Assay

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Alkaline phosphatase (ALP) activity was investigated using a p-nitrophenyl phosphate (pnPP) phosphatase kinetic assay [66 (link)] after homogenization of the constructs (n = 3) with a TissueLyser (5 min, 25 Hz). SIGMAFAST™ p-nitrophenyl phosphate tablets (Merck, Frankfurt, Germany) were used to prepare a substrate solution. Dephosphorylation of pnPP by ALP resulted in the production of a yellow compound that was measured for 30 min at 405 nm using an Infinite M200 Pro plate reader (Tecan, Männedorf, Switzerland). One measurement was performed every two minutes with a shaking step in between. Readings at 655 nm were subtracted to correct for non-specific background values. Calf intestinal alkaline phosphatase was used as standard.
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3

Osteogenic Differentiation of hSF-MSCs on PEKK/TCP

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Cell suspension, at a density of 2 × 105 cells/mL, was evenly added onto PEKK/TCP dropwise. Four groups were tested in vitro: (1) PEKK + OS (PEKK seeded with hSF-MSCs in osteogenic media), (2) PEKK + SF (PEKK seeded with hSF-MSCs in standard culture media), (3) TCP + OS (TCP seeded with hSF-MSCs in osteogenic media), and (4) TCP + SF (TCP seeded with hSF-MSCs in standard culture media). After 3 days of culture, the standard culture media in the OS groups was changed into an osteogenic media and renewed twice a week while the SF groups were renewed with standard medium at the same frequency. The cellular ALP activity was detected after 1, 4, 7, 14, and 21 days70 (link),71 (link). SigmaFast p-nitrophenyl phosphate tablets (Sigma) dissolved in deionized H2O was used with the cell lysate to detect the ALP level. The absorbance was read in a microplate reader at 405 nm (SpectraMax® M2). The ALP units were standardized to the cell number.
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4

Alkaline Phosphatase Activity Assay for Osteoblasts and MSCs

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The previously-described PIASPAC protocol was applied to assay the relative ALP activity in OBs and UC-MSCs with some modifications [40 (link),41 (link)]. Viable cells were seeded on the cell assay platform as droplets (OB: 1.4 × 104 cells/cm2, UC-MSC: 2.9 × 104 cells/cm2). After 7 days of culture, SIGMAFAST™ p-nitrophenyl phosphate tablets (N1891, SIGMA-ALDRICH, St. Louis, MO, USA) dissolved in water were added to the medium following the manufacturer’s protocol and incubated for 30 min. The absorbance of the supernatant was measured by an absorption plate reader (iMark™ Microplate Absorbance Reader) at 405 nm. Significant differences in the data between two conditions were determined by Student’s t-test.
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5

Quantifying ADAM17-Mediated Shedding in Cell Lines

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AP-HB-EGF HT1080, HeLa, and MCF-7 cells were reverse transfected with negative control, ADAM17 or PACS-2 siRNA in triplicate. After 72 hours, HT1080 cells were washed twice with serum-free medium (SFM) and stimulated with 400 nM PMA or SFM for 30 minutes at 37°C. The supernatant was transferred to 96-well plates and centrifuged. The cell layer was washed in PBS and lysed in 2xRIPA (100 mM Tris pH 7.4, 300 mM NaCl, 0.2% SDS, 2% Triton X-100, 1% sodium deoxycholate, 2 mM EDTA, with protease inhibitor cocktail). For colorimetric quantification of shedding, the cell-conditioned medium was mixed 1:1 with a 1 mg/ml solution of the alkaline phosphatase substrate 4-nitrophenyl (SIGMAFAST p-Nitrophenyl phosphate Tablets, Sigma-Aldrich) in technical duplicate. After incubation at 37°C in the dark, AP activity was quantified by measuring absorbance at 405 nm. Knockdown was verified by western blot.
Forty-eight hours after transfection, AP-HB-EGF HeLa were treated with 160 nM PMA or SFM for 45 minutes (as described18 (link)). Seventy-two hours after transfection, MCF-7 cells were stimulated with SFM, 160 nM PMA or 1 µM ionomycin for 30 minutes (as described27 (link)). Shedding of AP-HB-EGF and knockdown efficiency were analysed as above.
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6

Osteogenic Potential of MC3T3-E1 Cells

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ALP activity and staining were carried out to study the osteogenic potential of Murine preosteoblast MC3T3-E1 cells under experimental conditions, according to our previously published protocol (Behera et al., 2018 (link)) and others (Ming et al., 2013 (link); Y. K. Zhai et al., 2014 (link)). Briefly, MC3T3-E1 cells were grown in an osteogenic medium in 12-well culture plates. After 9 days of osteogenic induction, the cells were fixed with 4% formaldehyde and stained with SIGMAFAST™ p-nitrophenyl phosphate tablets (catalog No. N1891; Sigma-Aldrich, St. Louis, MO), and the images were then photographed. The number, total area, and relative intensities of ALP-positive colonies were counted and analyzed by using the image analysis software Image-Pro® plus 6.0.
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7

ELISA for Recombinant NTS-DBL1α Antibodies

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For the immune sera set, antibodies against the recombinant NTS-DBL1α were measured by ELISA as previously described [26 (link)]. Maxisorp plates (Nunc, Rosklid) were coated overnight at 4 °C with 1 μg of the recombinant antigen dissolved in 15 mM Na2CO3 and 35 mM NaHCO3 (pH 9.6). Plates were washed three times with PBS containing 0.1 % Tween 20 (PBST) and then blocked with 1 % bovine serum albumin (BSA) in PBST for 1 h at room temperature and subsequently washed three times with PBST. All samples were titrated by six serial dilutions in PBS to determine concentration dependency, and they were incubated in triplicate for 1 h at room temperature. Plates were washed three times with PBST, then bound IgG was measured by incubation for 1 h at room temperature with alkaline phosphatase-conjugated goat anti-human IgG (Sigma) diluted 1:1000 in PBS. Plates were washed three times with PBST and developed with SigmaFast p-nitrophenyl phosphate tablets (Sigma) for 15 min. The optical density (OD) was measured at 405 nm in an ELISA reader (Multiskan™ GO Microplate Spetrophotometer, Thermo Scientific). A control pool of six non-malaria exposed Swedish donors was included, as well as control wells without serum (background). Seropositivity was determined based on the 1:500 dilutions as the mean OD405 plus two standard deviations of the Swedish control pool.
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8

Quantifying Plasma C3c Complement Binding

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We used serial plasma dilutions (10%, 5%, 2.5%, 1.25%, and 0.75% in BBS) and pooled plasma of mice belonging to the same experimental group, incubating the samples for 15 min on mannan- (10 µg/mL) or acBSA-coated (25 µg/mL) plates prepared according to a procedure reported previously.18 (link) Plates were washed and incubated for 1 h 30 min at RT with a polyclonal anti-human-C3c antibody (Dako, A0062) diluted to 2.4 µg/mL in wash buffer. After washing, plates were incubated with an alkaline phosphatase-labeled goat anti-rabbit IgG antibody (Sigma A-3812) diluted to 1 µg/mL in wash buffer for 1 h 30 min at RT. After washing, the assay was developed by adding 100 µL of substrate solution (Sigma Fast p-Nitrophenyl Phosphate Tablets, Sigma). The absorbance at 405 nm was then measured using the Infinite M200 spectrofluorimeter managed by Magellan software (Tecan, CH).
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9

Serum IgE Quantification via ELISA

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Samples were incubated in the plate for ELISA (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) coated with a biotinylated anti-IgE mAb (for serum IgE) or TNP26-BSA (2.5 μg/ml) (for b4-SN). IgE concentration was quantified by staining with an AP-conjugated anti-IgE polyclonal Ab and SIGMAFAST™ p-Nitrophenyl phosphate Tablets (Sigma-Aldrich) and by measuring the absorbance at 405 nm.
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10

Quantifying ADAM17-Mediated Shedding in Cell Lines

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AP-HB-EGF HT1080, HeLa, and MCF-7 cells were reverse transfected with negative control, ADAM17 or PACS-2 siRNA in triplicate. After 72 hours, HT1080 cells were washed twice with serum-free medium (SFM) and stimulated with 400 nM PMA or SFM for 30 minutes at 37°C. The supernatant was transferred to 96-well plates and centrifuged. The cell layer was washed in PBS and lysed in 2xRIPA (100 mM Tris pH 7.4, 300 mM NaCl, 0.2% SDS, 2% Triton X-100, 1% sodium deoxycholate, 2 mM EDTA, with protease inhibitor cocktail). For colorimetric quantification of shedding, the cell-conditioned medium was mixed 1:1 with a 1 mg/ml solution of the alkaline phosphatase substrate 4-nitrophenyl (SIGMAFAST p-Nitrophenyl phosphate Tablets, Sigma-Aldrich) in technical duplicate. After incubation at 37°C in the dark, AP activity was quantified by measuring absorbance at 405 nm. Knockdown was verified by western blot.
Forty-eight hours after transfection, AP-HB-EGF HeLa were treated with 160 nM PMA or SFM for 45 minutes (as described18 (link)). Seventy-two hours after transfection, MCF-7 cells were stimulated with SFM, 160 nM PMA or 1 µM ionomycin for 30 minutes (as described27 (link)). Shedding of AP-HB-EGF and knockdown efficiency were analysed as above.
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