A tetrazolium method for non-specific alkaline phosphatase

McGadey, J

doi:10.1007/bf00305851

Cited by 269 publications

(66 citation statements)

References 4 publications

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“…All blots were washed extensively in TBST, and then in alkaline phosphatase buffer (100 mM NaCl, 5 mM MgCl 2 , 100 mM Tris, pH 9.5). Immunoreactivity of the secondary antibody was detected by 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT), as previously described (McGadey, 1970).…”

Section: Identification Of Ovoperoxidase Protein Targets Within the Fmentioning

confidence: 99%

Free-radical crosslinking of specific proteins alters the function of the egg extracellular matrix at fertilization

Wong

Wessel

2008

Development

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All animal embryos begin development by modifying the egg extracellular matrix. This protein-rich matrix protects against polyspermy, microbes and mechanical stress via enzyme-dependent transformations that alter the organization of its constituents. Using the sea urchin fertilization envelope, a well-defined extracellular structure formed within minutes of fertilization, we examine the mechanisms whereby limited permeability is established within this matrix. We find that the fertilization envelope acquires a barrier filtration of 40,000 daltons within minutes of insemination via a peroxidase-dependent mechanism, with dynamics that parallel requisite production of hydrogen peroxide by the zygote. To identify the molecular targets of this freeradical modification, we developed an in vivo technique to label and isolate the modified matrix components for mass spectrometry. This method revealed that four of the six major extracellular matrix components are selectively crosslinked, discriminating even sibling proteins from the same gene. Thus, specific free-radical chemistry is essential for establishing the embryonic microenvironment of early development.

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Section: Identification Of Ovoperoxidase Protein Targets Within the Fmentioning

confidence: 99%

Free-radical crosslinking of specific proteins alters the function of the egg extracellular matrix at fertilization

Wong

Wessel

2008

Development

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“…After hybridization to 10 ng/ml digoxigenin-labeled antisense probe, blots were treated with 2 g/ml RNase A, washed in 1ϫ SSC (150 mM NaCl and 15 mM sodium citrate), and probed for digoxigenin in maleate buffer (100 mM maleic acid, 185 mM NaCl, and 0.5% Tween 20, pH 8.0; Engler-Blum et al, 1993). Probes were detected using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium as described previously (McGadey, 1970).…”

Section: Rna Analysismentioning

confidence: 99%

Rendezvin: An Essential Gene Encoding Independent, Differentially Secreted Egg Proteins That Organize the Fertilization Envelope Proteome after Self-Association

Wong

Wessel

2006

MBoC

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Preventing polyspermy during animal fertilization relies on modifications to the egg's extracellular matrix. On fertilization in sea urchins, the contents of cortical granules are secreted and rapidly assemble into the egg's extracellular vitelline layer, forming the fertilization envelope, a proteinaceous structure that protects the zygote from subsequent sperm. Here, we document rendezvin, a gene whose transcript is differentially spliced to yield proteins destined for either cortical granules or the vitelline layer. These distinctly trafficked variants reunite after cortical granule secretion at fertilization. Together, they help coordinate assembly of the functional fertilization envelope, whose proteome is now defined in full.

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“…Each test zone was washed twice by adding 10 µL of PBS to the test zone, and then bringing the bottom of the paper-microzone plate in contact with a piece of blotting paper to absorb the excess wash buffer from the test zone. Finally, 3 µL of a solution of the colorimetric substrate for ALP (2.7-mM 5-bromo-4-chloro-3-indolyl phosphate disodium salt, 1.8-mM nitrotetrazolium blue chloride, 5-mM MgCl 2 , 100-mM NaCl, 0.05% Tween in 100-mM Tris buffer, pH 9.5) 21 was added to the test zone immediately after the washing step, and the color-producing enzymatic reaction was allowed to proceed for 30 minutes under ambient conditions. The test zone was scanned, and the intensity of the color was measured using ImageJ.…”

mentioning

confidence: 99%

Paper‐Based ELISA

et al. 2010

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# These authors contributed equally to this work.1 This paper describes enzyme-linked immunosorbent assays (ELISA) performed in a 96-microzone plate made out of paper (paper-based ELISA, or P-ELISA). ELISA is widely used in biochemical analyses, including immunoassays, food industry assays for food allergens, and toxicological assays. These assays are typically carried out in microtiter plates or small vials.1,2 ELISA combines the specificity of antibodies and the high-turnover catalysis by enzymes, to provide specificity and sensitivity. 1,2 We have recently described a 96-microzone paper plate-fabricated by patterning hydrophobic polymer in hydrophilic paper-as a platform for biochemical analysis. 3,4 Although microfluidic paper-based analytical devices (µPADs) were designed primarily to provide analytical capability at low cost in developing countries, [5][6][7] we expect that they will also be useful in applications such as point-of-care clinical analysis, military field operations, and others where high throughput, low volumes of sample, low cost, and robustness are important. 6,7 These devices have so far been prototyped using analyses of simple metabolites: glucose, protein, and certain enzymes. 8-10 P-ELISA combines the sensitivity and specificity of ELISA with the convenience, low cost and ease-of-use of paper-based platforms.Porous membranes, including nitrocellulose and filter paper, have been used for decades in dot-immunobinding assays (DIA). [8][9][10][11][12][13] Though DIAs are the simplest form of immunoassays on paper, they typically require one piece of nitrocellulose for each assay, the pieces of nitrocellulose have to be processed individually in Petri dishes, and the assays take several hours to complete. 9 Quantitative DIAs have been reported, 14 but DIAs are typically qualitative, and provide only "yes/no" results. 15 Traditional ELISA, usually performed in 96-well plates (fabricated by injection molding in plastic), is quantitative 2 and well-suited for high-throughput assays, but each assay requires large volumes (~20-200 µL) of analyte and reagents, the incubation and blocking steps are long (≥1 h per step, because the reagents must diffuse to the surface of the wells), and the results are quantified using a plate reader, typically an ~$20,000 instrument. 9,16Paper microzone plates for ELISA can have the same layout as plastic 96-wellplates, but each test zone requires only ~3 µL of sample, and the results can be measured using a desktop scanner, typically a ~$100 instrument. In addition, an entire P-ELISA can be completed in less than one hour. The ease of fabrication of paper microzone plates also opens opportunities for a wide range of non-standard formats, and customized connections to carry reagents between zones. To evaluate the feasibility of P-ELISA, and the potential advantages and disadvantages of P-ELISA and 96-well-plate-based ELISA,we developed a three-step procedure that i) immobilizes targeted antigens and then incubates them with their primary antibodies on a 96-microzo...

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A tetrazolium method for non-specific alkaline phosphatase

Cited by 269 publications

References 4 publications

Free-radical crosslinking of specific proteins alters the function of the egg extracellular matrix at fertilization

Free-radical crosslinking of specific proteins alters the function of the egg extracellular matrix at fertilization

Rendezvin: An Essential Gene Encoding Independent, Differentially Secreted Egg Proteins That Organize the Fertilization Envelope Proteome after Self-Association

Paper‐Based ELISA

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