Phosphatase substrate tablets

Manufactured by Merck Group

Phosphatase substrate tablets are a type of laboratory reagent used to detect and measure the activity of phosphatase enzymes. They provide a simple and reliable way to quantify the presence and activity of these enzymes in various biological samples.

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Lab products found in correlation

Immulon 2 plates, by Thermo Fisher Scientific (2 mentions) High binding 96 well plates, by Thermo Fisher Scientific (2 mentions) Rat anti mouse igm, by BD (1 mentions) Microplate manager software, by Bio-Rad (1 mentions) Xmark microplate spectrophotometer, by Bio-Rad (1 mentions) Phosphate buffer saline (pbs), by Merck Group (1 mentions) Immulon4 plates, by Thermo Fisher Scientific (1 mentions) Nonfat dry milk, by BD (1 mentions) Spectramax m2 plate reader, by Molecular Devices (1 mentions) Goat anti mouse igg alkaline phosphatase conjugate, by Merck Group (1 mentions) Pc bsa, by Biosearch Technologies (1 mentions) Bovine serum albumin (bsa), by Southern Biotech (1 mentions) Kc junior software, by Agilent Technologies (1 mentions) Goat serum, by Thermo Fisher Scientific (1 mentions) Nonfat dry milk, by Southern Biotech (1 mentions) Goat serum, by Southern Biotech (1 mentions) Dpbs t, by Merck Group (1 mentions) Non fat dry milk, by Bio-Rad (1 mentions)

10 protocols using phosphatase substrate tablets

Mouse IgM ELISA Quantification

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Immulon 2 plates (ThermoLabsystems, Pittsburgh, PA) were coated overnight at 4 °C with goat anti-mouse human adsorbed unlabeled IgM (Southern Biotech; 100 μl/well). Plates were washed, blocked with 3% BSA in PBS at 37 °C overnight, washed, and 100 μl/well of four two-fold dilutions of sera in PBS + 1% BSA were added starting at 1:2. Sample containing plates were incubated for 1 h at 37 °C and washed. Goat anti-mouse AP conjugated antibodies (Southern Biotech; 100 μl/well) were added for 1 h at 37 °C. Plates were washed and 100 µl of phosphatase substrate tablets (Sigma-Aldrich) dissolved in p-Nitrophenyl Phosphate, Disodium Salt (PNPP) buffer was added to wells. Absorbance was read at 405 nm on an xMark™ Microplate Spectrophotometer with the Microplate Manager™ Software (Bio-Rad, Hercules, CA). Standard curves were generated using serial dilutions of purified rat anti-mouse IgM (Clone II/41, BD), and the 4-parameter fit equation used to calculate sample concentrations.

Amato-Menker C.J., Hopen Q., Pettit A., Gandhi J., Hu G., Schafer R, & Franko J. (2024). XX sex chromosome complement modulates immune responses to heat-killed Streptococcus pneumoniae immunization in a microbiome-dependent manner. Biology of Sex Differences, 15, 21.

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ELISA for Antibody Detection

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Immulon4 plates (Thermo) were coated with E. coli-expressed full-length CSP recombinant protein at 1 μg/ml in Phosphate Buffer Saline (PBS, Sigma), incubated overnight at 4 °C, washed with wash buffer (0.05% Tween-20 in PBS), and blocked with 5% nonfat dry milk (BD Biosciences) for 2 h at RT. Plasma was added at 1:200 dilution and incubated for 2 h at RT. Alkaline-phosphatase labeled goat anti-human IgG (K + L) was added at 1:800 dilution and incubated for 2 h. Plates were developed with phosphatase substrate tablets (Sigma). After 20 min, OD was measured at 405 nm, with 650 nm correction, using a Spectramax^® M2 plate reader (Molecular Devices).

Finney O.C., Keitany G.J., Smithers H., Kaushansky A., Kappe S, & Wang R. (2014). Immunization with genetically attenuated P. falciparum parasites induces long-lived antibodies that efficiently block hepatocyte invasion by sporozoites. Vaccine, 32(19), 2135-2138.

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Quantifying HIV-1 Envelope-Specific Antibodies

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Sera were tested for HIV-1 gp140-specific and V2-specific Abs by ELISA on plates coated with gp140 protein or linear V2 peptide, respectively. HIV-1 strain Du172 gp140 protein was produced in baculovirus[19 (link)]. V2 peptide was based on a 19 amino acid immunodominant linear epitope within V2 of HIV-1 strain Du172: TTTEITDKKKKEYALFYKL (Genscript, Piscataway, NJ)[20 (link)]. Gp140-specific IgG was tested in 2-fold serum dilutions from 1:1000–1:16,000. V2-specific IgG was tested using 2-fold serum dilutions from 1:100–1:6400. Briefly, 96-well plates (Nunc-Immuno™) were coated overnight at 4°C with (A)gp140 protein (200ng/well) or (B)V2 peptide (400ng/well). Wells were blocked overnight at 4°C with (A)PBS/3% BSA/0.1% Tween-20 or (B)PBS/8% BSA/0.1% Tween-20. Duplicate samples and a standard were incubated at RT for (A)2h or (B)90 min. Bound IgG was detected with goat anti-mouse IgG alkaline phosphatase conjugate (Sigma-Aldrich) at (A)1:30,000 or (B)1:15,000. Bound enzyme was detected with phosphatase substrate tablets (Sigma-Aldrich) in DEA substrate buffer (KPL) by optical density (OD) at 405 nm. Gp140-specific IgG is reported as area under the curve (AUC) for 1:2000–1:16,000 dilution curves. V2-specific IgG is reported as OD at 1:100 dilution.

EMMER K.L., WIECZOREK L., TUYISHIME S., MOLNAR S., POLONIS V.R, & ERTL H.C. (2016). Antibody responses to prime-boost vaccination with an HIV-1 gp145 Env protein and chimpanzee adenovirus vectors expressing HIV-1 gp140. AIDS (London, England), 30(16), 2405-2414.

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Antibody Reactivity to Nuclear Antigens

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An ELISA was performed to assess the reactivity of the cloned antibodies to nuclear antigens. Quanta Lite ANA ELISA plates (Inova Diagnostics) were blocked with 3% FBS–PBS and incubated with purified antibodies at 10 μg/ml concentration. Alkaline phosphatase–coupled IgG was added, followed by phosphatase substrate tablets (Sigma). All incubations were carried out for 1 hour at 37°C, and the plates were washed with PBS–0.05% Tween.

Malkiel S., Jeganathan V., Wolfson S., Orduño N.M., Marasco E., Aranow C., Mackay M., Gregersen P.K, & Diamond B. (2016). Checkpoints for Autoreactive B Cells in the Peripheral Blood of Lupus Patients Assessed by Flow Cytometry. Arthritis & rheumatology (Hoboken, N.J.), 68(9), 2210-2220.

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ELISA for Anti-Ro52 Autoantibody Detection

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Furthermore, ELISA for anti-Ro52 autoantibodies was performed as previously described [42 (link)–45 (link)]. Briefly, high binding 96-well plates (Nunc) were coated with 1 μg of recombinant full-length Ro52 protein per well, diluted in carbonate buffer (pH 9.6). Before use, the plates were blocked with phosphate-buffered saline (PBS)/0.05% Tween/5% milk powder for 2 h. Sera diluted in Tween-PBS/1% milk powder were added and incubated for 2 h at room temperature. Autoantibody binding was detected with an alkaline phosphatase-conjugated rabbit anti-human IgG antibody 1:2000 (Dakopatts), which was incubated for 2 h at room temperature. Finally, binding was visualized using phosphatase substrate tablets (Sigma) and measured at 405 nm absorbance. A high anti-Ro52 titer serum was used as standard with serial dilutions for calculation of arbitrary units of antibody levels.

Ramírez Sepúlveda J.I., Kvarnström M., Brauner S., Baldini C, & Wahren-Herlenius M. (2017). Difference in clinical presentation between women and men in incident primary Sjögren’s syndrome. Biology of Sex Differences, 8, 16.

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P. berghei CSP Antibody Assay

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The repeat region of the P. berghei circumsporozoite protein (CSP) was a kind gift from Dr. Urszula Krych (WRAIR). The CSP peptide was coated at a concentration of 1ug/ml and incubated overnight at 4C. Serum collected from immunized mice prior to challenge (Study day 65) was used at a 1:100 dilution for the assay. After a 2-hour incubation, the plates were developed using alkaline phosphatase labeled goat anti-mouse IgG (Kirkegaard & Perry Labs, Inc. 1.0mg catalog # 075-1806) and Phosphatase substrate tablets (Sigma, cat. number S0942). Plates were read on a SpectraMax 340PC instrument.

Zaidi I., Diallo H., Conteh S., Robbins Y., Kolasny J., Orr-Gonzalez S., Carter D., Butler B., Lambert L., Brickley E., Morrison R., Sissoko M., Healy S.A., Lee Sim B.K., Doumbo O.K., Hoffman S.L, & Duffy P.E. (2017). γδ T cells are required for the induction of sterile immunity during irradiated sporozoite vaccinations. Journal of immunology (Baltimore, Md. : 1950), 199(11), 3781-3788.

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ELISA-Based Antibody-Secreting Cell Assay

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Millipore MultiScreen^® 96-well filter plates (Sigma-Aldrich) were coated with 50 μl phosphorylcholine (PC)-BSA (Biosearch Technologies, Petaluma, CA; 10 μg/ml) overnight at 4 °C. In subsequent steps, plates were washed with PBS + 0.01% Tween-20. Plates were blocked with 200 μl/well RPMI medium + 25% FBS for 2 h at 37 °C. Plates were washed and splenocytes (100 μl/well) added at 5 × 10⁶ cells/ml and 1 × 10⁶ cells/ml, each plated in triplicate. Plates were incubated for 4–6 h at 37 °C 5% CO₂. After washing, goat anti-mouse alkaline phosphatase (AP) conjugated IgM antibody (Southern Biotechnology Associates, Birmingham, AL), diluted 1/2000 in PBS + 1% BSA + 0.05% Tween-20, were added to the appropriate wells (100 μl/well). Plates were incubated overnight at 4 °C and washed. Phosphatase substrate tablets (Sigma-Aldrich) were dissolved in water and 100 μl added to each well. Color development was stopped by washing with water. The number of spots/well was counted using a dissection microscope (ZEISS, Dublin, CA). The number of ASC was calculated using the mean number of spots from triplicate wells. Mice demonstrating an average of less than 20 spots in the 5 × 10⁶ dilution were considered non-responders and not included in subsequent analyses. The number of ASC was normalized to 1 × 10⁶ splenic B220 + B cells as determined by flow cytometric analysis.

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Mouse Immunoglobulin ELISA Protocol

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Immulon 2 plates (ThermoLabsystems, Franklin, MA) were coated overnight at 4°C with 5 μg/ml PC-BSA (50 μl/well). Plates were washed, blocked with 3% BSA + PBS at 37°C for 2 hours, washed, and 100μl/well of eight twofold dilutions of sera in PBS +1% BSA were added starting at 1/400 for the IgG and IgM ELISAs and 1/50 for the IgG subclasses. Plates were incubated for 1 hour at 37°C and washed. Goat anti-mouse AP conjugated antibodies (100μl/well) were added for 1 hour at 37°C. Plates were washed, phosphatase substrate tablets (Sigma-Aldrich) dissolved in p-Nitrophenyl Phosphate, Disodium Salt (PNPP) buffer and added to wells. Absorbance was read at 405nm on a μQuant spectrophotometer using KCJunior software (Bio-Tek Instruments, Winooski, VT).

Holásková I., Franko J., Goodman R.L., Arnold A.P, & Schafer R. (2015). The XX sex chromosome complement is required in male and female mice for enhancement of immunity induced by exposure to 3,4-dichloropropionanilide. American journal of reproductive immunology (New York, N.Y. : 1989), 74(2), 136-147.

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EEEV E2 Glycoprotein ELISA Protocol

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Recombinant EEEV E2 glycoprotein (E3E2) (strain V105; IBT Bioservices) was diluted to 0.5 μg/mL in 1x D-PBS to coat 384-well ELISA plates (Thermo Fisher Scientific) at 25 μL/well and incubated at 4°C overnight. The plates were washed 3x with D-PBS-T (1x D-PBS + 0.05% Tween 20 [Cell Signaling Technology]) and blocked for 1 hour at room temperature with 25 μL/well blocking solution (2% non-fat dry milk (Bio-Rad), 2% goat serum (Gibco) in D-PBS-T). After blocking, the plates then were washed 3x with D-PBS-T and a volume of 10 to 25 μL/well of supernatant from each well containing EBV-transformed B cells or hybridoma cell lines was added. Plates were incubated for 2 hours at room temperature or overnight at 4°C. Plates then were washed 3x with D-PBS-T and a suspension of secondary antibodies (goat anti-human IgG-AP (Meridian Life Science) and goat anti-human IgA-AP [Southern Biotech]) at a 1:4,000 dilution in 1% blocking solution (1% non-fat dry milk, 1% goat serum) was added at 25 μL/well for 1 hour at room temperature. Alkaline phosphatase substrate solution (phosphatase substrate tablets (Sigma-Aldrich) in AP substrate buffer (1M Tris aminomethane and 30 mM MgCl₂) was added at 25 μL/well following plate washing 4x with D-PBS-T. Plates were incubated at room temperature in the dark for 1–2 hours and then read at an optical density of 405 nm with a plate reader.

Williamson L.E., Gilliland T J.r., Yadav P., Binshtein E., Bombardi R., Kose N., Nargi R.S., Sutton R.E., Durie C.L., Armstrong E., Carnahan R.H., Walker L.M., Kim A.S., Fox J.M., Diamond M.S., Ohi M.D., Klimstra W.B, & Crowe JE J.r. (2020). Human antibodies protect against aerosolized Eastern equine encephalitis virus infection. Cell, 183(7), 1884-1900.e23.

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Home-made ELISA for Anti-nuclear Antibodies

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The antibodies were tested using a home-made ELISA assay following the method outlined by Klauninger et al. [19] with minor variations. Briefly, high-binding 96-well plates (Nunc) were coated overnight (0.1 mg/well) with recombinant Ro52 and La antigens and with native Ro60 protein (Arotec Diagnostics Limited, Wellington, New Zealand) diluted in carbonate buffer (pH 9.6). Plates were blocked with 200 μL/well of phosphate-buffered saline PBS, 0.05% Tween-5% milk powder and sera were tested at 1:1000 dilution in PBS-0.05% Tween-1% milk powder. Bound antibodies were detected by affinity-purified alkaline phosphatase-conjugated goat anti-human IgG antibodies (Sigma-Aldrich) at 1:1000 dilution. Phosphatase substrate tablets (Sigma-Aldrich) dissolved in Mg-carbonate buffer (pH 9.8) were used as substrate. The absorbance was measured using a microplate reader at 405 nm, and the optical density was converted into arbitrary units using a high positive serum to build the three calibration curves at seven dilution points. The cutoff values were calculated as the 99th percentile of values obtained by testing the sera of 100 healthy women who were age-matched with the patients. All sera were also evaluated for anti-SSA/Ro52, /Ro60 and anti-SSB/La with an ELISA kit (Orgentec Diagnostika, Germany). The relevant results were compared with those obtained by the home-made ELISA.

Mattia E., Hoxha A., Tonello M., Favaro M., Del Ross T., Calligaro A., Ghirardello A, & Ruffatti A. (2018). Detection of autoantibodies to the p200-epitope of SSA/Ro52 antigen. A comparison of two laboratory assays. Clinical chemistry and laboratory medicine, 56(6).

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