ELISA plates were coated with 50 μl of plasma (1/200 dilution in PBS) from non-infected or infected mice at different times (21 to 124 dpi). After blocking with 1% bovine serum albumin (BSA), biotinylated Aptamer-29 (Apt-29) was added to each well and incubated for 1 hour at room temperature. Next, plates were washed three times with PBS and a Streptavidin-Alkaline phosphatase conjugate was added. Following a 30-minute incubation, plates were washed with PBS and the bound aptamers were detected using 4-Methyliumbeliferyl Phosphate (4-MUP, Sigma, MO). Fluorescence was read at 340 nm and emission was recorded at 440 nm, using a Spectra Max, M5 microplate reader (Molecular Devices, PA) [31 (link)].
4 mup
Manufactured by Merck Group
Sourced in United States
4-MUP is a laboratory reagent used in various biochemical and analytical applications. It is a fluorogenic substrate that can be used to detect and quantify the presence of specific enzymes or analytes in a sample. The core function of 4-MUP is to serve as a reliable tool for researchers and scientists in their experimental processes.
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Lab products found in correlation
4 protocols using 4 mup
1
Aptamer-based ELISA for Detecting Mouse Antibodies
2
Fluorometric Assay of Calcineurin Phosphatase
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Phosphatase activities of CaN were assayed fluorometrically using 4-methylumbelliferyl-phosphate (4-MUP, Sigma, St. Louis, MO, USA) as the substrate. The basal activity of CaN was determined using 200 µM substrate and 25 nM enzyme CaN in the buffer containing 50 mM Tris pH 8.6, 0.5 mM DTT, 1 mM MgCl2, and 0.3 mM CaCl2 at 25 °C. The CaM-dependent activation of the activity of CaN was measured by the addition of 25 nM CaM to the same reaction conditions described above. Fluorescence data were recorded using an Aminco Bowman (ABII) spectrofluorometer (Thermo Spectronic Inc., Rochester, NY, USA). The formation of the product, 4-MU, was monitored with excitation wavelength at 365 nm, and emission wavelength at 445 nm. Data were collected over a time course of 75 mins. A calibration curve was generated by measuring the fluorescence intensities of solutions containing various concentrations of 4-MU under the same assay conditions. The fluorescence intensities of the test samples were converted to the 4-MU concentration according to the calibration curve. The phosphatase activity of CaN is defined as µmol 4-MU produced per hour per µg CaN used.
3
Lysosomal Acid Lipase Activity Assay
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NSCs were seeded onto 10-cm dishes and grown as described for immunoblot analysis. On the day of harvest, cells were washed with DPBS and harvested in lysis buffer (0.5% Triton X-100 with protease inhibitor cocktail, Roche) with scraping. Cell lysates were pelleted at 13,200 × g for 5 min at 4 °C. The supernatant was removed and frozen at −80 °C until assayed. The LAL activity assay was modified from Guy et al. [19 (link)]. Briefly, 4-methylumbelliferyl palmitate (4-MUP, Cayman Chemicals) was dissolved to 400 μM in 1.45% (v/v) Triton X-100 with heating at 68 °C. cardiolipin sodium salt (Sigma-Aldrich) was dissolved in ethanol to 5 mg/mL. The substrate solution consisted of 133 μM 4-MUP, 0.65% Triton X-100 and 0.33 mg/mL cardiolipin in 67 mM acetate buffer pH 4.0. Reactions were initiated with the combination of 10 μL lysate (1 μg protein) and 30 μL of substrate solution. After 20 min at 37 °C, the reaction was terminated by adding 60 μL 5% perchloric acid, then 200 μL of 1 M sodium bicarbonate-sodium carbonate buffer pH 9.5. Fluorescence signal, with Ex/Em of 320/460 nm, was measured on an EnVision Multilabel Reader (PerkinElmer, Waltham, MA).
4
Quantitative Lysosomal Acid Lipase Assay
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Frozen liver and spleen samples were homogenized in LAL tissue extraction buffer (0.1 M sodium phosphate [pH 6.8], 1 mM EDTA, 0.02% sodium azide, 10 mM DTT, 0.5% NP-40). Protein concentrations were determined with the bicinchoninic acid assay (Pierce, Rockford, IL, USA), using BSA as the standard. LAL activity was determined using 4-methylumbelliferyl palmitate (4-MUP; Gold Biotechnology, St. Louis, MO, USA) as the substrate, as previously described.55 (link),56 (link) Briefly, 1 μg of protein or 1 μL serum was added to 0.345 mM substrate solution (0.345 mM 4-MUP, 90.9 mM sodium acetate [pH 4.0], 1% [v/v] Triton X-100 and 0.0325% [w/v] cardiolipin), and enzymatic reactions were performed in triplicate in the presence or absence of the LAL inhibitor Lalistat2 (Sigma Aldrich, St. Louis, MO, USA). Reactions were incubated at 37°C for 3 h in the dark. Reactions were terminated by adding 200 μL of 150 mM EDTA (pH 11.5). A standard curve was prepared ranging from 0–33.3 μM 4-methylumbelliferone (4-MU; Gold Biotechnology, St. Louis, MO, USA). Fluorescence was measured on a SpectraMax M2 plate reader (Molecular Devices, San Jose, CA, USA) using a 355-nm excitation filter and a 460-nm emission filter. LAL activity (pmol/min/μg) was calculated by subtracting the enzymatic activity of the inhibited reaction from that of the uninhibited reaction.
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