4 nitrophenyl phosphate disodium salt hexahydrate pnpp

Manufactured by Merck Group

Sourced in United States

4-Nitrophenyl phosphate disodium salt hexahydrate (pNPP) is a laboratory reagent commonly used as a substrate for the detection and quantification of various enzymes, particularly alkaline phosphatases. It is a colorless to white crystalline solid that undergoes a color change upon enzymatic hydrolysis, allowing for the spectrophotometric measurement of enzyme activity.

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Lab products found in correlation

Sodium chloride (nacl), by Avantor (1 mentions) Sodium citrate tribasic dehydrate, by Merck Group (1 mentions) Anhydrous sodium acetate, by Merck Group (1 mentions) Triton x 100, by Thermo Fisher Scientific (1 mentions) Trizma base, by Merck Group (1 mentions) Citric acid monohydrate, by Merck Group (1 mentions) Milli q water, by Merck Group (1 mentions) Anti mouse igg2, by BD (1 mentions) Alkaline phosphatase conjugated streptavidin, by Jackson ImmunoResearch (1 mentions) Purified mouse igg1, by BD (1 mentions) Purified mouse ige, by BD (1 mentions) Anti mouse igg1, by BD (1 mentions) Immulon4 plates, by Thermo Fisher Scientific (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions)

4 protocols using 4 nitrophenyl phosphate disodium salt hexahydrate pnpp

Biomimetic Extracellular Matrix Synthesis

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HEP sodium salt from porcine intestinal mucosa (≥180 USP units/mg, BioReagent, suitable for cell culture), PSS (Mw ~ 70,000 g/mol), sodium citrate tribasic dehydrate (ACS reagent), citric acid monohydrate (ACS reagent), 4-Nitrophenyl phosphate disodium salt hexahydrate (pNPP), sodium acetate anhydrous, and Trizma^® base were purchased from Sigma-Aldrich, St. Louis, MO, USA. Eagle’s Minimum Essential Medium (EMEM, ATCC^® 30-2003™) and BJ fibroblast cell (ATCC^® CRL-2522™) were purchased from ATCC, Manassas, VA, USA. Dulbecco’s phosphate-buffered saline (PBS, L0615) and Trypsin-EDTA 1X in PBS (L0940) were purchased from Dominique Dutscher, Bernolsheim, France. TA was obtained from Alfa Aesar, Haverhill, MA, USA. Purified type 1 COL was purchased from Symatese, Chaponost, France. Sodium dodecyl sulfate (SDS) was obtained from Prolabo, Warszawa, Poland. Sodium chloride was obtained from VWR, Radnor, PA, USA. Triton^® X-100 was purchased from Fisher Scientific, Waltham, MA, USA. Milli-Q water (18.2 MΩ·cm at 25 °C) supplied by Advantage A10 (MERCK) was used in the entire study. All the materials were used as received.

Chen Y.W., Iqbal M.H., Meyer F., Ball V, & Boulmedais F. (2023). Physical Chemistry Study of Collagen-Based Multilayer Films. Gels, 9(3), 192.

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Neutralization Assay of mAbs and Fabs

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The neutralization activity of mAbs and Fabs was investigated by pre- and post-virus attachment assays in 293TT cells. In the pre-attachment assay, PsVs were pre-incubated with mAb or Fabs at dilutions ranging from 66 nM-0.067 pM for 1 hour at 37 °C before adding to cells. For the post-attachment assay, PsVs were incubated with 293TT cells for 1 hour at 4 °C to allow for attachment. Cells were washed 1x with media prior to incubation with mAbs or Fabs. 72 hours later 30 ul of the cell culture supernatant was assayed with 4-Nitrophenyl phosphate disodium salt hexahydrate (pNPP) (Sigma) and the optical density was determined by absorbance spectrometry at OD405/490. The neutralization data was plotted at each antibody concentration and was used to determine the half-maximum neutralization titer.

Guan J., Bywaters S.M., Brendle S.A., Lee H., Ashley R.E., Makhov A.M., Conway J.F., Christensen N.D, & Hafenstein S. (2015). Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization. Virology, 483, 253-263.

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Enzyme-Linked Immunosorbent Assay for Viral Capsids

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PsV (500 ng) was bound to the wells of microtiter plates in PBS. The wells were washed with PBS/T to remove unbound particles and subsequently blocked with 5% dry milk protein in PBS/T. Next, the primary mAb (anti-L1 or anti-L2) was serially diluted and added to the wells in blocking buffer. A second primary mAb was added to the wells at a constant concentration (66.6 nM) followed by an anti-isotype specific alkaline phosphatase (AP) conjugated secondary antibody (Southern Biotech, Birmingham, AL, USA) at a 1:3000 dilution. The wells were developed in 4-Nitrophenyl phosphate disodium salt hexahydrate (pNPP) (Sigma-Aldrich, St. Louis, MO, USA) and the optical density was determined by absorbance spectrometry at optical density (OD) 405/450.

Bywaters S.M., Brendle S.A., Tossi K.P., Biryukov J., Meyers C, & Christensen N.D. (2017). Antibody Competition Reveals Surface Location of HPV L2 Minor Capsid Protein Residues 17–36. Viruses, 9(11), 336.

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Quantitative ELISA for Mouse Immunoglobulins

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Immulon 4 plates (Thermo Scientific, Waltham, MA) were coated with either purified mouse anti-IgE capture antibody (BD Biosciences), purified mouse anti-IgG1 (BD Pharmingen, Franklin Lakes, NJ) capture antibody or purified mouse anti-IgG2 (BD Pharmingen) capture antibody in 1X PBS and incubated overnight at 4°C. Then plates were washed with 1X PBS supplemented with 0.025% Tween 20 and blocked for 2 hours at 37 °C in 1X PBS supplemented with 5% BSA (Sigma Aldrich) and 0.05% Tween. Purified mouse IgE (BD Biosciences), purified mouse IgG1 (BD Biosciences) and purified mouse IgG2 (BD Biosciences) were used as standards. Blocked plates were washed with wash buffer and samples incubated for 2 hours at 37 °C. Plates were then washed and biotinylated anti-mouse IgE (BD Biosciences), anti-mouse IgG1 (BD Biosciences) or anti-mouse IgG2 (BD Biosciences) was added and incubated for 2 hours at 37 °C. Following a washing step, 1:1000 dilution of streptavidin conjugated alkaline phosphatase (Jackson Immunoresearch, West Grove, PA) was added to the plates and incubated for 1 hour at 37 °C. 4-Nitrophenyl phosphate disodium salt hexahydrate (PNPP) (Sigma Aldrich) was used as the substrate. Plates were read on an ELISA reader at 405/650.

Boyd A., Killoran K., Mitre E, & Nutman T.B. (2015). Pleural cavity Type 2 innate lymphoid cells precede Th2 expansion in murine Litomosoides sigmodontis infection. Experimental parasitology, 159, 118-126.

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