96‐well plates (NUNC, Maxisorp) were coated either with polyclonal α‐mouse IgM or IgG antibody (SouthernBiotech), respectively, and blocked with buffer containing 1% BSA. Dilutions of mouse IgM or IgG antibodies (SouthernBiotech) were used as standard. The concentration of IgM or IgG antibodies in the sera was determined by detection with alkaline phosphatase‐labeled α‐mouse IgM or IgG (Southern Biotech), respectively. P‐nitrophenylphosphate (Genaxxon) in diethanolamine buffer was added, and data were acquired at 405 nm using a Multiskan FC ELISA plate reader (Thermo Scientific). For detection of α‐HEL antibodies, sera were adjusted to an IgM concentration of 500 μg/ml and applied in dilution steps of 1:3 to plates coated with 10 μg/ml HEL. For detection of α‐TNP IgG antibodies, sera were adjusted to an IgG concentration of 1 μg/ml and applied in dilution steps of 1:3 to plates coated with 1 μg/ml TNP‐conjugated BSA.
P nitrophenyl phosphate
Manufactured by Thermo Fisher Scientific
Sourced in United States
P-nitrophenyl phosphate is a colorimetric substrate used for the detection and quantification of phosphatase enzyme activity. It is commonly used in various biochemical and molecular biology applications, such as enzyme-linked immunosorbent assays (ELISA).
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin alkaline phosphatase, by Thermo Fisher Scientific (3 mentions)
α mouse igm or igg antibody, by Southern Biotech (2 mentions)
α mouse igm or igg, by Southern Biotech (2 mentions)
Multiskan fc elisa plate reader, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Spectramax m5, by Molecular Devices (2 mentions)
Stempro osteogenic induction medium, by Thermo Fisher Scientific (2 mentions)
5 bromo 4 chloro 3 indolyl phosphate nitro blue tetrazolium, by Merck Group (2 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Flexstation 3 microplate reader, by Molecular Devices (1 mentions)
Lysozyme, by R&D Systems (1 mentions)
Trypsin, by neoFroxx (1 mentions)
Alkaline phosphatase, by Thermo Fisher Scientific (1 mentions)
Lambda exonuclease, by New England Biolabs (1 mentions)
Radioimmunoprecipitation assay (ripa), by Solarbio (1 mentions)
T4 ligase, by New England Biolabs (1 mentions)
Na3vo4, by Merck Group (1 mentions)
Thrombin, by Prolytix (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
L cysteine, by Sinopharm (1 mentions)
31 protocols using p nitrophenyl phosphate
1
Quantification of Mouse Antibody Isotypes
2
Alkaline Phosphatase Assay for Anti-estrogenic Activity
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EXAMPLE 14
Method for performing the alkaline phosphatase (AP) assay. ECC-1 cells were trypsinized and resuspended in hormone-depleted media and plated at a density of 15k cells per well into a 96-well plate for at least 4 hours. Cells were treated with antiestrogens for 3 days and plates were subsequently frozen at −80° C. Thawed plates were incubated with a chromogenic substrate of AP, p-nitrophenyl phosphate (Thermo Fisher Scientific), for 40 minutes at 40° C. and absorbances were read at 405 nm. AP activity was normalized to the activity of E2 alone. This assay was shown to correlate with the in vivo studies comparing uterine wet weight in ovariectomized rats following treatment with a number of anti-estrogens. A representative result for induction of AP activity in uterine cells (% E2) is shown below in tabular form:
3
Osteoblast Differentiation by ALP Activity
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Osteoblast cell differentiation was estimated by ALP activity. Osteoblast cells were lysed in a buffer solution containing 0.05% Triton X-100, 1.0% Tris, and 6.0% NaCl (w/v in deionized water, pH 10.0. All the chemicals were procured from Sigma Aldrich (St. Louis, MI, USA). A volume of 60 µL of scaffold specimen solution was added to 50 µL of 0.07% p-nitrophenylphosphate (w/v, Thermo Fisher, Waltham, MA, USA) in amino methyl propanol (AMP, Acros Organics, Pittsburgh, PA, USA) buffer and then the resulting solution was placed in an incubator for 2 h at 37 °C. The absorbance was recorded at 400 nm. ALP activities were normalized to the ALP activity/µg of the entire DNA content [27 (link)].
4
Quantification of ALP Activity in hMSCs
5
Enzyme-Linked Immunosorbent Assay for Mayaro Virus
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Optimal target quantities along with dilution of patient serum and secondary antibody were determined by checkerboard titration. After optimization, Dx-MAYV-M chimera was used at 300 ng/well in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6) to sensitize high-binding 96-well microplates (Jet Biofil, Guangzhou, China) overnight at 4 °C. Microplates were rinsed and blocked with TBSM (TBS with 1% fat-free dry milk) for 1 h at 37 °C and washed with TBST before adding patient serum samples (100 μl, diluted 1:150 in TBS). Plates were incubated for 90 min at 37 °C, washed in TBST, and then incubated with alkaline phosphatase-conjugated goat anti-human IgM (1:20,000) for 90 min at 37 °C. Next, plates were washed five times before the addition of 100 μl of PNPP (p-nitrophenyl phosphate, 1 mg/ml, Thermo Fisher, USA). After a 15 min incubation at room temperature in the dark, the absorbance at 405 nm was measured in a FlexStation-3 microplate reader (Molecular Devices, San José, CA, U.S.A.).
6
Sensitive Immunoassay Detection with Exonuclease
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Alkaline phosphatase (ALP, 1000 U/mL) and p-nitrophenyl phosphate (PNPP) were purchased from Thermo Fisher Scientific Inc (Shanghai, China). Lambda exonuclease (λ exo, 5000 U/mL) and T4 ligase were obtained from New England Biolabs (Ipswich, MA, USA). Thrombin was supplied from Haematologic Technologies (Essex Junction, VT, USA). Lysozyme was purchased from R&D Systems (Minneapolis, MN, USA). Human serum albumin (HSA) was bought from Tagene Biotechnology Co., Ltd. (Xiamen, China). Proteinase K was purchased from BBI Science (Shanghai, China). Trypsin was purchased from Biofroxx (Einhausen, Germany). Na3VO4, human IgG and anti-human IgG-coated ELISA plates were purchased from Sigma-Aldrich (St. Louis, MO, USA). L-cysteine was bought from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Human serum was obtained from Affiliated Chenggong Hospital (Xiamen University). Streptavidin (SA) Sepharose beads were purchased from GE Healthcare (Chicago, IL, USA). ALP-labeled goat anti-human IgG was purchased from Abcam (Cambridge, MA, USA). RIPA and PMSF were bought from Solarbio life sciences (Beijing, China). All other chemicals were analytical reagent grade and used without further purification. All oligonucleotides used in this study were synthesized and HPLC purified by Sangon Biotechnology Co., Ltd. (Shanghai, China) and sequences are listed in Table S1 .
7
Assessing Osteoblastic Formation and Mineralization of BMSCs
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The osteoblastic formation and mineralization abilities of BMSCs after osteogenesis for 7 days were detected by ALP staining. To assess the osteoblastic formation, the BMSCs seeded in 24-well plates (Nest, China) were continuously maintained in osteogenic induction medium (Cyagen Biosciences, China) under differentiating conditions. After osteogenesis for continuous 7 days, the mineralization of the calcium nodules was observed through ALP staining by using ALP staining kits (CTCC Bioscience, China). The stained BMSCs were subsequently visualized using an invert light microscope (Nikon, Japan), followed by the capture of representative images. To visualize the osteoblastic activity of BMSCs, the stained mineralization was dissolved by 10 mM p-nitrophenyl phosphate (Thermo, USA), and then the OD values at 420 nm were measured and calculated.
8
Alkaline Phosphatase Assay for Antiestrogen Activity
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Example 14
Method for performing the alkaline phosphatase (AP) assay. ECC-1 cells were trypsinized and resuspended in hormone-depleted media and plated at a density of 15 k cells per well into a 96-well plate for at least 4 hours. Cells were treated with antiestrogens for 3 days and plates were subsequently frozen at −80° C. Thawed plates were incubated with a chromogenic substrate of AP, p-nitrophenyl phosphate (Thermo Fisher Scientific), for 40 minutes at 40° C. and absorbances were read at 405 nm. AP activity was normalized to the activity of E2 alone. This assay was shown to correlate with the in vivo studies comparing uterine wet weight in ovariectomized rats following treatment with a number of anti-estrogens. A representative result for induction of AP activity in uterine cells (% E2) is shown below in tabular form:
9
Alkaline Phosphatase Assay for Anti-Estrogen Activity
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Example 14
Method for performing the alkaline phosphatase (AP) assay. ECC-1 cells were trypsinized and resuspended in hormone-depleted media and plated at a density of 15 k cells per well into a 96-well plate for at least 4 hours. Cells were treated with antiestrogens for 3 days and plates were subsequently frozen at −80° C. Thawed plates were incubated with a chromogenic substrate of AP, p-nitrophenyl phosphate (Thermo Fisher Scientific), for 40 minutes at 40° C. and absorbances were read at 405 nm. AP activity was normalized to the activity of E2 alone. This assay was shown to correlate with the in vivo studies comparing uterine wet weight in ovariectomized rats following treatment with a number of anti-estrogens. A representative result for induction of AP activity in uterine cells (% E2) is shown below in tabular form:
10
Alkaline Phosphatase Assay for Evaluating Anti-Estrogen Activity
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Example 14
Method for performing the alkaline phosphatase (AP) assay. ECC-1 cells were trypsinized and resuspended in hormone-depleted media and plated at a density of 15 k cells per well into a 96-well plate for at least 4 hours. Cells were treated with antiestrogens for 3 days and plates were subsequently frozen at −80° C. Thawed plates were incubated with a chromogenic substrate of AP, p-nitrophenyl phosphate (Thermo Fisher Scientific), for 40 minutes at 40° C. and absorbances were read at 405 nm. AP activity was normalized to the activity of E2 alone. This assay was shown to correlate with the in vivo studies comparing uterine wet weight in ovariectomized rats following treatment with a number of anti-estrogens. A representative result for induction of AP activity in uterine cells (% E2) is shown below in tabular form:
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