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Index / Bioassays / pNPP Phosphatase Assay Kit / Product Detail : POPN-500 pNPP Phosphatase Assay Kit
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#POPN-500 pNPP Phosphatase Assay Kit

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  Price : 218   EUR
247   USD
169   GBP
916   Zloty
29200   JPY
1683   NOK
1803   SEK
246   CHF

Product name : pNPP Phosphatase Assay Kit

Catalog number : POPN-500

Quantity: 500 Tests

Availability: Yes

Supplier name : Bioassays

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More Details about

For quantitative determination of phosphatase activity.
Method: OD405nm.
Samples: protein phosphatases.
Species: all.
Procedure: 30 min.
Size: 500 tests.
Detection limit: 3 ng.

DESCRIPTION
Para-nitrophenyl phosphate (pNPP) is a chromogenic substrate for most
phosphatases such as alkaline phosphatases, acid phosphatases, protein
tyrosine phosphatases and serine/threonine phosphatases. The reaction
yields para-nitrophenol, which becomes an intense yellow soluble product
under alkaline conditions and can be conveniently measured at 405 nm on
a spectrophotometer.
phosphatase
p-Nitrophenyl phosphate p-nitrophenol + phosphate
This homogeneous "mix-and-measure" assay involves simply adding a
single reagent to the phosphatase and measuring the product formation
on an absorbance reader. The assay can be conveniently performed in
cuvettes, tubes or multi-well plates at either room temperature or 37°C.
KEY FEATURES
High sensitivity and wide linear range. The detection limit is generally 3
ng phosphatase or below.
Homogeneous and simple procedure. No wash or reagent transfer
steps are involved. The assay can be completed within 30 minutes.
Robust and amenable to HTS. All reagents are compatible with highthroughput
liquid handling instruments.
APPLICATIONS
Enzyme Activity Assay and Quality Control for phosphatase
production.
Characterization of Kinetics of phosphatase reaction.
Drug Discovery: high-throughput screen for phosphatase inhibitors.
KIT CONTENTS
Catalog # Size (assays) Reagent Assay Buffer Stop Solution
POPN-500 500 280 μL 25 mL 25 mL
POPN-01K 1,000 550 μL 50 mL 50 mL
POPN-HTS >10k customized customized customized
Storage conditions. The kit is shipped at ambient temperature. Store
Reagent at -20°C and other components at 4°C. Shelf life: 12 months after
receipt.
Precautions: reagents are for research use only. Normal precautions for
laboratory reagents should be exercised while using the reagents. Please
refer to Material Safety Data Sheet for detailed information.

 

Sample Type: cells

Species: human

 

References: Monick, MM et al (2006). Active ERK Contributes to Protein Translation by Preventing JNK-Dependent Inhibition of Protein Phosphatase. J. Immunol. 177: 1636–1645. 

Pubmed ID: 16849472

Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=16849472

Abstract: Human alveolar macrophages, central to immune responses in the lung, are unique in that they have an extended life span in contrast to precursor monocytes. We have shown previously that the ERK MAPK (ERK) pathway is constitutively active in human alveolar macrophages and contributes to the prolonged survival of these cells. We hypothesized that ERK maintains survival, in part, by positively regulating protein translation. In support of this hypothesis, we have found novel links among ERK, JNK, protein phosphatase 1 (PP1), and the eukaryotic initiation factor (eIF) 2alpha. eIF2alpha is active when hypophosphorylated and is essential for initiation of protein translation (delivery of initiator tRNA charged with methionine to the ribosome). Using [(35)S]methionine labeling, we found that ERK inhibition significantly decreased protein translation rates in alveolar macrophages. Decreased protein translation resulted from phosphorylation (and inactivation) of eIF2alpha. We found that ERK inhibition increased JNK activity. JNK in turn inactivated (via phosphorylation) PP1, the phosphatase responsible for maintaining the hypophosphorylated state of eIF2alpha. As a composite, our data demonstrate that in human alveolar macrophages, constitutive ERK activity positively regulates protein translation via the following novel pathway: active ERK inhibits JNK, leading to activation of PP1alpha, eIF2alpha dephosphorylation, and translation initiation. This new role for ERK in alveolar macrophage homeostasis may help to explain the survival characteristic of these cells within their unique high oxygen and stress microenvironment.

PMID: 16849472 [PubMed - indexed for MEDLINE]

 

Sample Type: mesenchymal stem cells

Species: human

 

References: Cucchiarini M et al (2011). Metabolic activities and chondrogenic differentiation of human mesenchymal stem cells following recombinant adeno-associated virus-mediated gene transfer and overexpression of fibroblast

Pubmed ID: 21417714

Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=21417714

Abstract: The genetic manipulation of bone marrow-derived mesenchymal stem cells (MSCs) is an attractive approach to produce therapeutic platforms for settings that aim at restoring articular cartilage defects. Here, we examined the effects of recombinant adeno-associated virus (rAAV)-mediated overexpression of human fibroblast growth factor 2 (hFGF-2), a mitogenic factor also known to influence MSC differentiation, upon the proliferative and chondrogenic activities of human MSCs (hMSCs) in a three-dimensional environment that supports chondrogenesis in vitro. Prolonged, significant FGF-2 synthesis was noted in rAAV-hFGF-2-transduced monolayer and aggregate cultures of hMSCs, leading to enhanced, dose-dependent cell proliferation compared with control treatments (rAAV-lacZ transduction and absence of vector administration). Chondrogenic differentiation (proteoglycans, type-II collagen, and SOX9 expression) was successfully achieved in all types of aggregates, without significant difference between conditions. Most remarkably, application of rAAV-hFGF-2 reduced the expression of type-I and type-X collagen, possibly due to increased levels of matrix metalloproteinase-13, a key matrix-degrading enzyme. FGF-2 overexpression also decreased mineralization and the expression of osteogenic markers such as alkaline phosphatase, with diminished levels of RUNX-2, a transcription factor for osteoblast-related genes. Altogether, the present findings show the ability of rAAV-mediated FGF-2 gene transfer to expand hMSCs with an advantageous differentiation potential for future, indirect therapeutic approaches that aim at treating articular cartilage defects in vivo.

PMID: 21417714 [PubMed - in process]

 

 

Sample Type: follicles, eggs

Species: marine teleost

 

References: Sawaguchi S et al (2006). Identification of two forms of vitellogenin-derived phosvitin and elucidation of their fate and roles during oocyte maturation in the barfin flounder, Verasper moseri. Zoolog Sci. 23(11):1021-9.

Pubmed ID: 17189915

Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=17189915

Abstract: A new method for visualizing small and multiple phosvitins (Pvs) in oocytes from a marine teleost was developed by a combination of gel filtration, alkaline phosphatase treatment, and SDS-PAGE followed by silver staining. Three distinct Pv polypeptides having molecular masses of 15 kDa, 8 kDa, and 7 kDa were visualized in vitellogenic follicle extract of barfin flounder, Verasper moseri. N-terminal amino acid sequencing identified two different N-termini that fell into the PvA (7 kDa) and PvB (15 kDa and 8 kDa) groups, which were derived from two forms of vitellogenin (Vg), VgA and VgB, respectively. Analysis of time-course change in phosphorus-rich peaks of gel chromatography fractions of follicle extracts from different maturational stages demonstrated a rapid degradation of Pvs during mid-phase of oocyte maturation. Quantitative analysis of free amino acids in maturing follicles revealed an increment of serine content but not of phosphoserine, indicating the occurrence of dephosphorylation concomitant with Pv degradation. Measurement of phosphatase activity in follicles and eggs at different maturational stages demonstrated a significant activation of phosphatase especially under acidic conditions. This suggested that Pv degradation and dephosphorylation are regulated by changes in ooplasm pH during oocyte maturation. Our results also suggested that the Pvs in barfin flounder vitellogenic oocytes bind to much lower amounts of calcium and magnesium than those of masu salmon, Oncorhynchus masou. This indicates that the Pvs in the barfin flounder, a marine teleost spawning its eggs in seawater, do not play a role in the transport and deposition of calcium and magnesium into oocytes.

PMID: 17189915 [PubMed - indexed for MEDLINE]

 

Sample Type: cell supernatant

Species: mouse

 

References: Nakano Y (2007). Novel function of DUSP14/MKP6 (dual specific phosphatase 14) as a nonspecific regulatory molecule for delayed-type hypersensitivity. British J. Dermatology 156 (5): 848–860.

Pubmed ID: 17263825

Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=17263825

Abstract:
BACKGROUND:
Nonspecific unresponsive states of delayed-type hypersensitivity (DTH) to unrelated antigens are induced in mice by a single administration of hapten. In these studies, we found a unique regulatory mechanism of contact hypersensitivity (CHS) mediated by nonspecific suppressor factor (NSF) induced by the intravenous injection of hapten-conjugated syngeneic spleen cells. NSF is a approximately 45-kDa protein released from the macrophage-like suppressor cells and binds selectively to dendritic cells (DCs). Moreover, NSF-treated DCs release a second approximately 20-kDa NSF (NSF(int)).

OBJECTIVES:
To try and identify NSF and characterize its function.

METHOD
The suppressor activity was evaluated by inhibition of the passive transfer of CHS by the effector cells sensitized with hapten and the antigen-presenting cell (APC) activity of hapten-primed draining lymph node cells (DLNCs) to induce CHS. NSF-containing supernatants obtained from the culture of spleen cells from mice that had been injected intravenously with oxazolone-conjugated syngeneic spleen cells 7 days before were prepared and purified with a Green A dye-affinity column, DEAE column and Sephacryl S-200 column. Then, samples of molecular mass of approximately 45 kDa were separated by native-PAGE (polyacrylamide gel electrophoresis) and nonreducing sodium dodecyl sulphate (SDS)-PAGE. After confirming the suppressor activity of proteins of approximately 45 kDa separated by native-PAGE, samples were separated by nonreducing SDS-PAGE, transferred onto polyvinylidene difluoride membranes and analysed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

RESULTS:
Proteins of approximately 45 kDa eluted from a Sephacryl S-200 column and the slice of native-PAGE gel exhibited the strong suppressor activity. Analyses using MALDI-TOF mass spectrometry and MASCOT algorithm of the protein bands around 45 kDa separated by nonreducing SDS-PAGE identified NSF as a 22.5-kDa protein, dual specific phosphatase 14/MAP-kinase phophatase-6 (DUSP14/MKP6), which functions as a negative regulator of the MAP-kinase signalling. Western blot analyses revealed that recombinant DUSP14 (rDUSP14) exists as the mixture of 22.5-kDa monomer and 45-kDa dimer under nonreducing conditions, and monomers under reducing conditions. Treatment with rDUSP14 at 4 degrees C for 2 h suppressed the ability of effector cells to transfer CHS dose dependently and the APC function of DLNCs to induce CHS. Epicutaneous application of rDUSP14 immediately after challenge inhibited the subsequent CHS expression. rDUSP14 was bound specifically by major histocompatibility complex class II (Ia)-positive spleen cells (presumably DCs). The suppressor activity of NSF was neutralized by anti-DUSP14 monoclonal antibody. Expression of DUSP14 mRNA in the spleen was upregulated parallel to the unresponsive state induced by hapten-conjugated cells. NSF, NSF(int) and rDUSP14 exhibited the phosphatase activity towards p-nitrophenyl phosphate in vitro as alkaline phosphatase.

CONCLUSIONS:
These studies indicate for the first time that NSF is a dimer of DUSP14 secreted by macrophage-like suppressor cells by stimulation with hapten-conjugated cells and exerts a regulatory function on CHS through DCs as a secreted phosphatase.

PMID: 17263825 [PubMed - indexed for MEDLINE]

 

Sample Type: cells

Species: bacteria

 

References: Lee SW et al (2008). The Xanthomonas oryzae pv. oryzae PhoPQ two-component system is required for AvrXA21 activity, hrpG expression, and virulence. J Bacteriol. 190(6):2183-97.

Pubmed ID: 18203830

Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=18203830

Abstract: The rice pathogen recognition receptor, XA21, confers resistance to Xanthomonas oryzae pv. oryzae strains producing the type one system-secreted molecule, AvrXA21. X. oryzae pv. oryzae requires a regulatory two-component system (TCS) called RaxRH to regulate expression of eight rax (required for AvrXA21 activity) genes and to sense population cell density. To identify other key components in this critical regulatory circuit, we assayed proteins expressed in a raxR gene knockout strain. This survey led to the identification of the phoP gene encoding a response regulator that is up-regulated in the raxR knockout strain. Next we generated a phoP knockout strain and found it to be impaired in X. oryzae pv. oryzae virulence and no longer able to activate the response regulator HrpG (hypersensitive reaction and pathogenicity G) in response to low levels of Ca2+. The impaired virulence of the phoP knockout strain can be partially complemented by constitutive expression of hrpG, indicating that PhoP controls a key aspect of X. oryzae pv. oryzae virulence through regulation of hrpG. A gene encoding the cognate putative histidine protein kinase, phoQ, was also isolated. Growth curve analysis revealed that AvrXA21 activity is impaired in a phoQ knockout strain as reflected by enhanced growth of this strain in rice lines carrying XA21. These results suggest that the X. oryzae pv. oryzae PhoPQ TCS functions in virulence and in the production of AvrXA21 in partnership with RaxRH.

PMID: 18203830 [PubMed - indexed for MEDLINE] PMCID: PMC2258857

 

Sample Type: serum

Species: zebrafish

 

References: Olsen AS et al (2010). Limb regeneration is impaired in an adult zebrafish model of diabetes mellitus. Wound Repair Regen. 18(5):532-42.

Pubmed ID: 20840523

Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=20840523

Abstract: The zebrafish (Danio rerio) is an established model organism for the study of developmental processes, human disease, and tissue regeneration. We report that limb regeneration is severely impaired in our newly developed adult zebrafish model of type I diabetes mellitus. Intraperitoneal streptozocin injection of adult, wild-type zebrafish results in a sustained hyperglycemic state as determined by elevated fasting blood glucose values and increased glycation of serum protein. Serum insulin levels are also decreased and pancreas immunohistochemisty revealed a decreased amount of insulin signal in hyperglycemic fish. Additionally, the diabetic complications of retinal thinning and glomerular basement membrane thickening (early signs of retinopathy and nephropathy) resulting from the hyperglycemic state were evident in streptozocin-injected fish at 3 weeks. Most significantly, limb regeneration, following caudal fin amputation, is severely impaired in diabetic zebrafish and nonspecific toxic effects outside the pancreas were not found to contribute to impaired limb regeneration. This experimental system using adult zebrafish facilitates a broad spectrum of genetic and molecular approaches to study regeneration in the diabetic background.

© 2010 by the Wound Healing Society.

PMID: 20840523 [PubMed - indexed for MEDLINE] PMCID: PMC2941236

 

Sample Type: intestinal tissue

Species: chicken

 

References: Thanissery R et al (2010). Evaluation of the efficacy of yeast extract in reducing intestinal Clostridium perfringens levels in broiler chickens. Poult Sci. 89(11):2380-8.

Pubmed ID: 20952700

Pubmed link: http://www.ncbi.nlm.nih.gov/pubmed?term=20952700

Abstract: The etiological agent of necrotic enteritis is Clostridium perfringens. Traditionally, necrotic enteritis is controlled with in-feed antibiotics. However, increasing consumer demand for drug-free poultry has fostered the search for nonantibiotic alternatives. Yeast extract contain nucleotides that are immunomodulatory and also essential for cellular functions. An experiment was conducted to evaluate the efficacy of NuPro yeast extract (Alltech Inc., Nicholasville, KY) in reducing intestinal C. perfringens levels in broiler chickens. One hundred ninety-two 1-d-old male broiler chicks were obtained and randomly assigned to 6 treatments in a battery cage trial. Treatment 1 consisted of chicks fed a corn-soybean meal basal diet (BD) without added bacitracin methylene disalicylate or NuPro. Treatment 2 consisted of chicks fed BD into which bacitracin methylene disalicylate was added at 0.055 g/kg. Treatment 3 consisted of chicks fed BD supplemented with NuPro at a 2% level for the first 10 d of the experiment. Treatments 4 (PX), 5, and 6 (PN) consisted of chicks that were challenged with 3 mL of the C. perfringens inoculum (~10(7) cfu/mL) on d 14, 15, and 16 of the experiment and fed diets similar to treatments 1, 2, and 3, respectively. On d 1 and 7 postchallenge, intestinal C. perfringens levels, lesion scores, and alkaline phosphatase activity were assessed. On d 1 postchallenge, C. perfringens level in treatment 5 (2.09 log(10) cfu/g) was lower (P < 0.05) compared with the PX treatment (4.71 log(10) cfu/g) but similar to the PN treatment (2.98 log(10) cfu/g). A similar trend was observed on d 7 postchallenge. NuPro supplementation enhanced alkaline phosphatase activity (P < 0.05) in C. perfringens-challenged chicks and appeared to reduce intestinal lesion scores. Although dietary supplementation of NuPro in the PN treatment reduced C. perfringens levels by 1.73 and 0.68 log(10) cfu/g compared with the PX treatment on d 1 and 7 postchallenge, respectively, these reductions were not significant. Extending the period of NuPro supplementation beyond the first 10 d of life should be considered for achieving significant reduction in intestinal C. perfringensg levels.

PMID: 20952700 [PubMed - indexed for MEDLINE]

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