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#POPB-500 PiBlue™ Phosphate Assay Kit
Product name : PiBlue™ Phosphate Assay Kit
Catalog number : POPB-500
Supplier name : Bioassays
Data sheet: Ask more or other datasheet now !
More Details about
For sensitive and high-throughput phosphate determination.
Method: OD620nm (malachite green).
Samples: enzyme reactions with free phosphate releases.
Procedure: 30 min.
Size: 500 tests.
Detection limit: 0.4 μM.
The PiBlueTM Phosphate Assay Kit is based on a proprietary formulation of
the malachite green dye. The PiBlueTM reagent forms a blue colored
complex with free orthophosphate. The rapid color formation from the
reaction can be conveniently measured on a spectrophotometer (600 - 660
nm) or on a plate reader. The non-radioactive colorimetric assay kits have
been optimized to offer superior sensitivity and prolonged shelf life. The
assay is simple and fast, involving a single addition step for phosphate
determination. Assays can be performed in tubes, cuvettes or multi-well
plates. The assays can be conveniently executed in 96-well plates for highthroughput
screening of enzyme inhibitors.
Reagent very stable. Due to our innovative formulation, no precipitation of
reagent occurs. Therefore no filtration of reagent is needed prior to assays,
as is often required with other commercial kits.
High sensitivity and wide detection range: detection of as little of 20
pmoles of phosphate and useful range between 0.4 μM and 50 μM
Fast and convenient: single reagent “mix-and-measure” assay allows
quantitation of free phosphate within 30 minutes.
Compatible with routine laboratory and HTS formats: assays can be
performed in tubes, cuvettes or microplates, on spectrophotometers and
Robust and amenable to HTS: Z’ factors of 0.7 to 0.9 are observed in 96-
well plates. Can be readily automated on HTS liquid handling systems.
Phosphatase Assays: liberation of phosphate from peptide, protein or
small molecule substrate.
Lipase Assays: liberation of phosphate from phospholipids
Nucleoside Triphosphatase Assays: liberation of phosphate from
nucleoside triphosphates (ATP, GTP, TTP, CTP etc).
Quantitation of Phosphate in phospholipids, proteins and DNAs, etc.
Drug Discovery: high-throughput screen for phosphatase inhibitors.
Storage conditions. The kit is shipped at ambient temperature. The
PiBlueTM Reagent and standard is stable for 12 months when stored at 4°C.
This protocol can be downloaded online at www.bioassaysys.com.
Precautions: reagents are for research use only. This reagent contains
0.44 M sulfuric acid. Normal precautions for laboratory reagents should be
exercised while using the reagents.
Sample Type: cytosolic fraction
References: Kang HY et al (2008). Altered TNSALP expression and phosphate regulation contribute to reduced mineralization in mice lacking androgen receptor. Mol Cell Biol.28(24):7354-67.
Pubmed ID: 18838539
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=18838539
Abstract: While androgen receptor (AR)-deficient mice developed osteopenia in endochondral bones due to the high bone turnover with increased bone resorption by osteoclasts, little is known about the mechanism of intramembranous bone loss contributed by AR in osteoblasts. Here, we discovered a dramatic decrease in the area of calcification, new bone, and the number of osteocytes in calvaria from AR-deficient mice related to a reduction in mineralization caused, in part, by the diminished activity of AR-deficient osteoblasts. Enforced AR expression in differentiated osteoblasts boosts mineralization while knockdown of AR expression prevents androgen-induced mineralization. We identified the tissue-nonspecific alkaline phosphatase (TNSALP) and several members of small integrin binding ligand N-linked glycoprotein (SIBLING) gene family as androgen target genes required for AR-mediated bone formation. We show that inorganic phosphate (P(i)) levels and TNSALP activity increased in response to androgen/AR and P(i) signals increase the expression and translocation of AR. The ectopic expression of TNSALP or P(i) partially rescued the bone loss due to AR deficiency. Thus, androgen/AR signaling plays an essential role in bone formation by coordinating the expression of genes associated with phosphate regulation.
PMID: 18838539 [PubMed - indexed for MEDLINE] PMCID: PMC2593437
Sample Type: intracellular posphate
References: Hough TA et al (2007). Novel mouse model of autosomal semidominant adult ypophosphatasia has a splice site mutation in the tissue nonspecific alkaline phosphatase gene Akp2. J Bone Miner Res. 22(9):1397-407.
Pubmed ID: 17539739
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=17539739
Abstract: Deactivating mutations in the TNSALP gene cause HPP. Akp2(-/-) mice model severe infantile HPP, but there is no model for the relatively mild adult form. Here we report on mice with an induced mutation in Akp2 that affects splicing. The phenotype of homozygotes mirror aspects of the adult form of HPP.
Hypophosphatasia (HPP) is a clinically varied skeletal disorder resulting from deficiency of tissue nonspecific alkaline phosphatase (TNSALP). Mice lacking Akp2 model infantile HPP characterized by skeletal hypomineralization, impaired growth, seizures, and perinatal mortality. No animal model exists to study the less severe forms of the disease that typically present in later life.
MATERIALS AND METHODS:
N-ethyl-N-nitrosourea (ENU) mutagenesis was used to generate mouse models of human disease. A mouse with low plasma alkaline phosphatase (ALP) activity was identified by our clinical chemistry screen. Its offspring were used for inheritance studies and subjected to biochemical, histological, and radiological phenotyping. DNA was extracted for mapping and osteoblasts harvested for functional studies.
We showed semidominant inheritance of the low ALP phenotype and mapped the underlying point mutation to Akp2. Affected offspring bear the splice site mutation 862 + 5G>A-a hypomorphic allele named Akp2(Hpp). The same mutation has been reported in a patient. Akp2(Hpp/+) mice have approximately 50% of normal plasma ALP but display no other biochemical or skeletal abnormalities. Unlike Akp2(-/-) mice, Akp2(Hpp/Hpp) mice have normal initial skeletal development and growth, a normal lifespan and do not have seizures. TNSALP is low but detectable in Akp2(Hpp/Hpp) plasma. Osteoblasts display approximately 10% of normal ALP activity and reduced intracellular inorganic phosphate levels, yet are capable of normal mineralization in vitro. TNSALP substrates are significantly elevated in urine (inorganic pyrophosphate and phosphoethanolamine) and plasma (pyridoxal 5'-phosphate), whereas plasma inorganic pyrophosphate levels are normal. Akp2(Hpp/Hpp) mice develop late-onset skeletal disease, notably defective endochondral ossification and bone mineralization that leads to arthropathies of knees and shoulders.
Akp2(Hpp/Hpp) mice mirror a number of clinical features of the human adult form of HPP. These mice provide for the first time an animal model of late onset HPP that will be valuable in future mechanistic studies and for the evaluation of therapies such as those aimed at HPP.
PMID:17539739 [PubMed - indexed for MEDLINE]
Sample Type: lipid Pi
References: López-Vales R et al (2010). Fenretinide promotes functional recovery and tissue protection after spinal cord contusion injury in mice. J Neurosci. 30(9):3220-6.
Pubmed ID: 20203181
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=20203181
Abstract: The inflammatory response is thought to contribute to secondary damage after spinal cord injury (SCI). Polyunsaturated fatty acids (PUFAs) play an important role in the onset and resolution of inflammation. Arachidonic acid (AA), an omega-6 PUFA, contributes to the initiation of inflammatory responses, whereas docosahexaenoic acid (DHA), an omega-3 PUFA, has antiinflammatory effects. Therefore, decreasing AA and increasing DHA levels after SCI might be expected to attenuate inflammation after SCI and promote tissue protection and functional recovery. We show here that daily oral administration of fenretinide after spinal cord contusion injury led to a significant decrease in AA and an increase in DHA levels in plasma and injured spinal cord tissue. This was accompanied by a significant reduction in tissue damage and improvement in locomotor recovery. Fenretinide also reduced the expression of proinflammatory genes and the levels of oxidative stress markers after SCI. In addition, in vitro studies demonstrated that fenretinide reduced TNF-alpha (tumor necrosis factor-alpha) expression by reactive microglia. These results demonstrate that fenretinide treatment after SCI can reduce inflammation and tissue damage in the spinal cord and improve locomotor recovery. These beneficial effects may be mediated via the ability of fenretinide to modulate PUFA homeostasis. Since fenretinide is currently in clinical trials for the treatment of cancers, this drug might be a good candidate for the treatment of acute SCI in humans.
PMID: 20203181 [PubMed - indexed for MEDLINE]
Sample Type: cellular extract
References: Polewski MD et al (2010). Inorganic pyrophosphatase induces type I collagen in osteoblasts. Bone 46(1):81-90.
Pubmed ID: 19733704
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=19733704
The physiologic selectivity of calcification in bone tissue reflects selective co-expression by osteoblasts of fibrillar collagen I and of tissue nonspecific alkaline phosphatase (TNAP), which hydrolyzes the calcification inhibitor pyrophosphate (PP(i)) and generates phosphate (P(i)). Humans and mice deficient in the PP(i)-generating ecto-enzyme NPP1 demonstrate soft tissue calcification, occurring at sites of extracellular matrix expansion. Significantly, the function in osteoblasts of cytosolic inorganic pyrophosphatase (abbreviated iPP(i)ase), which generates P(i) via PP(i) hydrolysis with neutral pH optimum, remains unknown. We assessed iPP(i)ase in Enpp1(-/-) and wild type (WT) mouse osteoblasts and we tested the hypothesis that iPP(i)ase regulates collagen I expression.
We treated mouse calvarial osteoblasts with ascorbate and beta-glycerol phosphate to promote calcification, and we assessed cytosolic P(i) and PP(i) levels, sodium-dependent P(i) uptake, Pit-1 P(i) co-transporter expression, and iPP(i)ase and TNAP activity and expression. We also assessed the function of transfected Ppa1 in osteoblasts.
Inorganic pyrophosphatase but not TNAP was elevated in Enpp1(-/-) calvariae in situ. Cultured primary Enpp1(-/-) calvarial osteoblasts demonstrated increased calcification despite flat TNAP activity rather than physiologic TNAP up-regulation seen in WT osteoblasts. Despite decreased cytosolic PP(i) in early culture, Enpp1(-/-) osteoblasts maintained cytosolic P(i) levels comparable to WT osteoblasts, in association with increased iPP(i)ase, enhanced sodium-dependent P(i) uptake and expression of Pit-1, and markedly increased collagen I synthesis. Suppression of collagen synthesis in Enpp1(-/-) osteoblasts using 3,4-dehydroproline markedly suppressed calcification. Last, transfection of Ppa1 in WT osteoblasts increased cytosolic P(i) and decreased cytosolic but not extracellular PP(i), and induced both collagen I synthesis and calcification.
Increased iPP(i)ase is associated with "P(i) hunger" and increased calcification by NPP1-deficient osteoblasts. Furthermore, iPP(i)ase induces collagen I at the levels of mRNA expression and synthesis and, unlike TNAP, stimulates calcification by osteoblasts without reducing the extracellular concentration of the hydroxyapatite crystal inhibitor PP(i).
Published by Elsevier Inc.
PMID: 19733704 [PubMed - indexed for MEDLINE] PMCID: PMC2818162
Sample Type: blood, semen
References: Oborna I et al (2010). Increased lipid peroxidation and abnormal fatty acid profiles in seminal and blood plasma of normozoospermic males from infertile couples. Hum Reprod. 25(2):308-16.
Pubmed ID: 19942612
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=19942612
Abstract: Reactive oxygen species (ROS) are essential for sperm function. However, excessive ROS production can impair sperm function and might be a factor contributing to male infertility.
We investigated the levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) as well as lipid peroxidation, as represented by thiobarbituric acid reactive species (TBARS), in blood and seminal plasma of 38 normozoospermic males from infertile couples (NSI-males), compared with that of 17 fertile volunteers (FV-males).
TBARS levels in blood and seminal plasma were higher in NSI-males than in FV-males (P < 0.0002, P < 0.0003, respectively), as were AA levels (P < 0.0003, P < 0.00004, respectively). On the contrary, the blood and seminal plasma levels of DHA were lower in NSI-males than in FV-males (P < 0.02 and P < 0.05, respectively). The AA/DHA ratios in blood and seminal plasma were higher in NSI-males than in FV-males (P < 0.003, P < 0.0007, respectively). Significant correlations between seminal and blood plasma levels of TBARS (P < 0.0001, r = 0.548), AA (P < 0.0001, r = 0.571) and DHA (P < 0.0001, r = 0.506) were found.
Our data provide new insight into lipid metabolism in male infertility and indicate that systemic oxidative stress resulting in increased lipid peroxidation and an altered fatty acid profile may be, at least in part, responsible for infertility even in normozoospermic males.
PMID: 19942612 [PubMed - indexed for MEDLINE]
Sample Type: phosphatases
References: Lau KH et al (2006). An osteoclastic protein-tyrosine phosphatase is a potential positive regulator of the c-Src protein-tyrosine kinase activity: a mediator of osteoclast activity. J Cell Biochem. 97(5):940-55.
Pubmed ID: 16267838
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=16267838
Abstract: This study tested the hypothesis that an osteoclastic protein-tyrosine phosphatase, PTP-oc, enhances osteoclast activity through c-Src activation. The effects of several resorption activators and inhibitors on PTP-oc expression, resorption activity, and c-Src activation were determined in rabbit osteoclasts. PTP-oc expression was assayed with immunoblots and semi-quantitative RT-PCR. Osteoclastic activity was determined by the resorption pit assay; and c-Src activation was monitored by P-tyr527 (PY527) dephosphorylation, and in vitro kinase assay. Treatment of osteoclasts with PTH, PGE2, 1,25(OH)2D3, IL-1, but not RANKL or IL-6, significantly stimulated resorption activity, increased PTP-oc mRNA and protein levels, and reduced c-Src PY527 level with corresponding activation of c-Src protein-tyrosine kinase activity. The PTP-oc antisense phosphorothioated oligo treatment blocked the basal and IL-1alpha-mediated, but not RANKL-mediated, resorption activity of isolated osteoclasts. The antisense oligo treatment also significantly reduced the average depth of resorption pits created by rabbit osteoclasts under basal conditions. Calcitonin and alendondrate, significantly reduced resorption activity and PTP-oc expression, and increased c-Src PY527 with corresponding reduction in its PTK activity. The cellular PTP-oc protein level correlated with the resorption activity. Among the various signaling proteins co-immunoprecipitated with PTP-oc, the resorption effectors caused corresponding changes in the tyrosyl phosphorylation level of only c-Src. The GST-PTP-oc fusion protein dephosphorylated PY-527-containing c-Src peptide in time- and dose-dependent manner in vitro. In summary, (1) PTP-oc is regulated in part at transcriptional level, (2) upregulation of PTP-oc in osteoclasts led to c-Src activation, and (3) PY527 of c-Src may be a cellular substrate of PTP-oc. These findings are consistent with the hypothesis that PTP-oc is a positive regulator of c-Src in osteoclasts.
(c) 2005 Wiley-Liss, Inc.
PMID: 16267838 [PubMed - indexed for MEDLINE]
Sample Type: Na+/K+-ATPase extract
References: Cramp RL et al (2009). Ups and downs of intestinal function with prolonged fasting during aestivation in the burrowing frog, Cyclorana alboguttata. J Exp Biol. 212(22):3656-63.
Pubmed ID: 19880727
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=19880727
Abstract: Although green striped burrowing frogs (Cyclorana alboguttata) experience large reductions in the mass and absorptive surface area of the small intestine (SI) during aestivation, little is known about how this may affect the functional capacity of the SI. We examined changes in the function (l-proline uptake rate and capacity) and metabolism of the SI (in vitro oxygen consumption, Na(+)/K(+)-ATPase activity and abundance) of C. alboguttata following 6 months of aestivation. l-Proline uptake rate was significantly higher in aestivating frogs, but overall uptake capacity was lower than in active frogs. Total SI oxygen consumption rate (V(O(2))) was also lower in aestivating frogs, despite no difference in mass-specific V(O(2)). The proportion of intestinal V(O(2)) associated with Na(+)/K(+)-ATPase activity and protein synthesis was equivalent between active and aestivating frogs, suggesting these processes were unaffected by aestivation. Indeed, the activity of Na(+)/K(+)-ATPase transporters in the SI of aestivating frogs was not different from that of active animals. Aestivating frogs maintained Na(+)/K(+)-ATPase activity, despite experiencing a reduction in the density of Na(+)/K(+)-ATPase transporters, by increasing the molecular activity of the remaining pumps to 2-3 times that of active frogs. These results show that functionality of the SI is maintained at the cellular level, potentially facilitating the reclamation of nutrients from the intestinal lumen while in aestivation. Despite this, the functional capacity of the SI in aestivating C. alboguttata is significantly reduced due to a reduction in tissue mass, helping frogs to conserve energy while in aestivation.
PMID: 19880727 [PubMed - indexed for MEDLINE]
Sample Type: ATPase extract
References: Huxtable AG et al (2009). Tripartite purinergic modulation of central respiratory networks during perinatal development: the influence of ATP, ectonucleotidases, and ATP metabolites. J Neurosci. 29(47):14713-25
Pubmed ID: 19940166
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=19940166
Abstract: ATP released during hypoxia from the ventrolateral medulla activates purinergic receptors (P2Rs) to attenuate the secondary hypoxic depression of breathing by a mechanism that likely involves a P2Y(1)R-mediated excitation of preBötzinger complex (preBötC) inspiratory rhythm-generating networks. In this study, we used rhythmically active in vitro preparations from embryonic and postnatal rats and ATP microinjection into the rostral ventral respiratory group (rVRG)/preBötC to reveal that these networks are sensitive to ATP when rhythm emerges at embryonic day 17 (E17). The peak frequency elicited by ATP at E19 and postnatally was the same ( approximately 45 bursts/min), but relative sensitivity was threefold greater at E19, reflecting a lower baseline frequency (5.6 +/- 0.9 vs 19.0 +/- 1.3 bursts/min). Combining microinjection techniques with ATP biosensors revealed that ATP concentration in the rVRG/preBötC falls rapidly as a result of active processes and closely correlates with inspiratory frequency. A phosphate assay established that preBötC-containing tissue punches degrade ATP at rates that increase perinatally. Thus, the agonist profile [ATP/ADP/adenosine (ADO)] produced after ATP release in the rVRG/preBötC will change perinatally. Electrophysiology further established that the ATP metabolite ADP is excitatory and that, in fetal but not postnatal animals, ADO at A(1) receptors exerts a tonic depressive action on rhythm, whereas A(1) antagonists extend the excitatory action of ATP on inspiratory rhythm. These data demonstrate that ATP is a potent excitatory modulator of the rVRG/preBötC inspiratory network from the time it becomes active and that ATP actions are determined by a dynamic interaction between the actions of ATP at P2 receptors, ectonucleotidases that degrade ATP, and ATP metabolites on P2Y and P1 receptors.
PMID: 19940166 [PubMed - indexed for MEDLINE]
Sample Type: lipid
References: Chen X et al (2007). The isolation and structure of membrane lipid rafts from rat brain. Biochimie 89(2):192-6.
Pubmed ID: 16935406
Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=16935406
Abstract: The action of detergents in the isolation of detergent-resistant membrane fractions from rat brain is reported. Triton X-100 treatment of whole rat brain homogenate at 4 degrees C produced detergent-resistant membranes with a density of 1.07g/ml compared with Brij96 where the density of the membrane was only 1.05g/ml. The DRM fractions isolated using Triton X-100 are considerably heavier than those isolated from homogenates treated with Brij96. The major polar lipid composition of DRMs derived from Brij96 treated homogenates have a higher proportion of aminophospholipids compared with choline phospholipids than Triton X-100 derived DRMs; this may indicate that DRMs from Brij96 treated homogenates are more closely related to the parent membrane in lipid composition. Solubilization by Triton X-100 at higher temperatures resulted in the appearance of a second detergent-resistant membrane fraction distinctly lighter in density than the membrane recovered at density 1.07g/ml. Analysis of phospholipid composition of the brain homogenate during detergent treatment for up to 30min at 37 degrees C showed a decreasing proportion of sphingomyelin. Treatment of homogenates at 37 degrees C appears to activate phospholipases/sphingomyelinases that may alter the lipid content of isolated DRMs. The presence of K+/Mg2+ with Brij96 treatment results in DRM fractions with significantly thicker bilayers and of larger vesicle diameter than DRMs isolated from either Triton X-100 or Brij96 treated homogenates in the absence of cations.
PMID: 16935406 [PubMed - indexed for MEDLINE]
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WP1004: Kit Receptor Signaling Pathway
WP1028: Pentose Phosphate Pathway
WP1121: Kit Receptor Signaling Pathway
WP1147: Pentose Phosphate Pathway
WP122: Pentose Phosphate Pathway
WP1231: Pentose Phosphate Pathway
WP134: Pentose Phosphate Pathway
WP1341: Kit Receptor Signaling Pathway
WP147: Kit Receptor Signaling Pathway
WP1493: Carbon assimilation C4 pathway
WP1657: Glycerolipid metabolism
WP1658: Glycerophospholipid metabolism
WP1664: Inositol phosphate metabolism
WP1684: Pentose phosphate pathway
WP2185: Purine metabolism
WP220: Ribose and Deoxyribose Phosphate Metabolism
WP2212: Methylerythritol phosphate pathway
WP228: Deoxyribose phosphate metabolism
WP2340: Thiamine (vitamin B1) biosynthesis and salvage
WP2341: vitamin B1 (thiamin) biosynthesis and salvage pathway
WP260: Glucose-1-phosphate metabolism
WP282: Pentose Phosphate Pathway
WP312: Pentose Phosphate Pathway
WP369: Pentose Phosphate Pathway
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