Western Analysis Using the Chemiluminescent Alkaline Phosphatase Substrate CSPD

2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.prot4256
Author(s):  
Peter Gillespie
Keyword(s):  
Alkaline Phosphatase ◽  
Western Analysis ◽  
Alkaline Phosphatase Substrate ◽  
Phosphatase Substrate

Detection of salmonellas by DNA hybridization with a fluorescent alkaline phosphatase substrate

1992 ◽  
Vol 72 (5) ◽  
pp. 393-399 ◽  
Author(s):  
R. J. Cano ◽  
M.J. Torres ◽  
R.E. Klem ◽  
J.C. Palomares ◽  
J. Casadesus
Keyword(s):  
Alkaline Phosphatase ◽  
Dna Hybridization ◽  
Alkaline Phosphatase Substrate ◽  
Phosphatase Substrate

scholarly journals Novel Live Alkaline Phosphatase Substrate for Identification of Pluripotent Stem Cells

2012 ◽  
Vol 8 (3) ◽  
pp. 1021-1029 ◽  
Author(s):  
Upinder Singh ◽  
Rene H. Quintanilla ◽  
Scott Grecian ◽  
Kyle R. Gee ◽  
Mahendra S. Rao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Pluripotent Stem Cells ◽  
Alkaline Phosphatase Substrate ◽  
Phosphatase Substrate

scholarly journals Use of a new fluorogenic phosphatase substrate in immunohistochemical applications.

1995 ◽  
Vol 43 (1) ◽  
pp. 77-83 ◽  
Author(s):  
K D Larison ◽  
R BreMiller ◽  
K S Wells ◽  
I Clements ◽  
R P Haugland
Keyword(s):  
Alkaline Phosphatase ◽  
Stokes Shift ◽  
High Magnification ◽  
Adult Zebrafish ◽  
Large Stokes Shift ◽  
Retinal Tissue ◽  
Bright Yellow ◽  
Immunohistochemical Techniques ◽  
Phosphatase Substrate

We used the phosphatase substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6- chloro-4-[3H]-quinazolinone, with standard alkaline phosphatase-mediated immunohistochemical techniques, to visualize a number of antibodies that bind to adult zebrafish retinal tissue. This compound, known as the ELF (enzyme-labeled-fluorescence) phosphatase substrate, produces a precipitate that fluoresces at approximately 500-580 nm (bright yellow-green). We show that the precipitated product from the ELF phosphatase substrate has a number of characteristics that make it superior to fluorescein-labeled secondary reagents. The staining produced with the ELF substrate is much more photostable than that produced by fluorescein-labeled secondary reagents, thus allowing time to examine, focus, and photograph the ELF-labeled tissue under high magnification. Moreover, the ELF precipitate exhibits a Stokes shift of greater than 100 nm, a characteristic that has enabled us to overcome the problem of distinguishing signal from background in this autofluorescent tissue. In addition, we show that the ELF product's large Stokes shift makes the ELF substrate ideal for multicolor applications.

scholarly journals The ELF-97 Alkaline Phosphatase Substrate Provides a Bright, Photostable, Fluorescent Signal Amplification Method for FISH

1997 ◽  
Vol 45 (3) ◽  
pp. 345-357 ◽  
Author(s):  
Violette B. Paragas ◽  
Yu-Zhong Zhang ◽  
Richard P. Haugland ◽  
Victoria L. Singer
Keyword(s):  
Alkaline Phosphatase ◽  
Cultured Cells ◽  
Mdck Cells ◽  
Blot Hybridization ◽  
Stokes Shift ◽  
Southern Blot Hybridization ◽  
Zebrafish Embryos ◽  
Tissue Sections ◽  
Whole Mount ◽  
Phosphatase Substrate

We used the ELF-97 (Enzyme-Labeled Fluorescence) phosphatase substrate, 2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone, with alkaline phosphatase conjugates of streptavidin and appropriate antibodies to amplify signals from biotinylated and haptenylated hybridization probes. The dephosphorylated product, ELF-97 alcohol, is a bright yellow-green fluorescent precipitate optimally excited at ∼360 nm, with emission centered at ∼530 nm. This large Stokes shift allows ELF-97 signals to be easily distinguished from sample autofluorescence and signals arising from counterstains or other fluorophores. The ELF-97 precipitate was extremely photostable compared to fluorescein, allowing multiple photographic exposures of samples without significant signal intensity loss. For RNA in situ hybridization, labeling was specific and localized well to targets in cultured cells, tissue sections, and whole-mount zebrafish embryos. ELF-97 signals developed in seconds to minutes and were easily distinguished from pigmented tissues or cells, unlike those obtained using colorimetric substrates. We used the substrate with singly biotinylated short oligonucleotides to detect actin mRNA in MDCK cells and actin and β-galactosidase mRNA in LacZ+ mouse fibroblasts. We also used a biotinylated cDNA, complementary to the mRNA encoded by the constant region of the T-cell receptor β-chain, to specifically identify T-cells in mouse lymph node tissue sections. With digoxigenin-labeled probes, we detected several developmentally expressed mRNAs in whole-mount zebrafish embryos. Hybridization to centromere repeat regions in human metaphase and interphase chromosomes was also detected; ELF-97 signals were manyfold brighter than signals obtained with fluorescein conjugates. Finally, Southern blot hybridization using singly labeled oligonucleotide probes yielded a sensitivity similar to that obtained with radioactivity. (J Histochem Cytochem 45:345–357, 1997)

scholarly journals Chronic Kidney Disease-Induced Arterial Media Calcification in Rats Prevented by Tissue Non-Specific Alkaline Phosphatase Substrate Supplementation Rather Than Inhibition of the Enzyme

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1138
Author(s):  
Britt Opdebeeck ◽  
Ellen Neven ◽  
José Luis Millán ◽  
Anthony B. Pinkerton ◽  
Patrick C. D’Haese ◽  
...  
Keyword(s):  
Chronic Kidney Disease ◽  
Alkaline Phosphatase ◽  
Kidney Disease ◽  
Inorganic Phosphate ◽  
Bone Mineralization ◽  
Control Diet ◽  
High Serum ◽  
Specific Alkaline Phosphatase ◽  
Metabolic Defects ◽  
Alkaline Phosphatase Substrate

Patients with chronic kidney disease (CKD) suffer from arterial media calcification and a disturbed bone metabolism. Tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes the calcification inhibitor pyrophosphate (PPi) into inorganic phosphate (Pi) and thereby stimulates arterial media calcification as well as physiological bone mineralization. This study investigates whether the TNAP inhibitor SBI-425, PPi or the combination of both inhibit arterial media calcification in an 0.75% adenine rat model of CKD. Treatments started with the induction of CKD, including (i) rats with normal renal function (control diet) treated with vehicle and CKD rats treated with either (ii) vehicle, (iii) 10 mg/kg/day SBI-425, (iv) 120 µmol/kg/day PPi and (v) 120 µmol/kg/day PPi and 10 mg/kg/day SBI-425. All CKD groups developed a stable chronic renal failure reflected by hyperphosphatemia, hypocalcemia and high serum creatinine levels. CKD induced arterial media calcification and bone metabolic defects. All treatments, except for SBI-425 alone, blocked CKD-related arterial media calcification. More important, SBI-425 alone and in combination with PPi increased osteoid area pointing to a less efficient bone mineralization. Clearly, potential side effects on bone mineralization will need to be assessed in any clinical trial aimed at modifying the Pi/PPi ratio in CKD patients who already suffer from a compromised bone status.

Interrelationship of the cellular distribution and secretion of regular structure (RS) protein in Bacillus licheniformis nm 105

1992 ◽  
Vol 50 (1) ◽  
pp. 712-713
Author(s):  
Xiaorong Zhu ◽  
Richard McVeigh ◽  
Bijan K. Ghosh
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Licheniformis ◽  
Self Assembly ◽  
Gel Electrophoresis ◽  
Culture Supernatant ◽  
Regular Structure ◽  
The Self ◽  
Cellular Distribution ◽  
Structural Protein ◽  
Laboratory Cultures

A mutant of Bacillus licheniformis 749/C, NM 105 exhibits some notable properties, e.g., arrest of alkaline phosphatase secretion and overexpression and hypersecretion of RS protein. Although RS is known to be widely distributed in many microbes, it is rarely found, with a few exceptions, in laboratory cultures of microorganisms. RS protein is a structural protein and has the unusual properties to form aggregate. This characteristic may have been responsible for the self assembly of RS into regular tetragonal structures. Another uncommon characteristic of RS is that enhanced synthesis and secretion which occurs when the cells cease to grow. Assembled RS protein with a tetragonal structure is not seen inside cells at any stage of cell growth including cells in the stationary phase of growth. Gel electrophoresis of the culture supernatant shows a very large amount of RS protein in the stationary culture of the B. licheniformis. It seems, Therefore, that the RS protein is cotranslationally secreted and self assembled on the envelope surface.

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