Application

• For quantitative determination of phosphatase enzyme activity.

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 high-throughput liquid handling instruments.

Method

• OD405nm

Samples

• Protein phosphatases

Species

• All

Procedure

• 30 min

Size

• 500 tests

Detection Limit

• 3 ng

Shelf Life

• 12 months

More Details

• p-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. 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°

I would like to run a positive control, to compare to her data. Will the Sigma enzyme (catalog # P6772, bovine alkaline phosphatase) work with your assay kit as a positive control? (i.e. pNPP substrate + assay buffer = units of activity as compared to the known units of activity in the Sigma enzyme)

The Sigma enzyme should work with our assay kit. As indicated in our assay protocol, the customer needs to prepare their own enzyme buffer (100 mM Tris-HCl (pH 8.6), 10 mM MgCl₂ or similar buffers) to raise the pH to 8.6 for an alkaline phosphatase.

Does your pNPP kit measure the units of activity or does it measure the moles of pNPP liberated/ minute?

Out kit measures the moles of pNPP liberated per minute as a unit for enzyme activity.

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 growth factor 2. Tissue Eng Part A. 17(15-16):1921-33. Assay: Phosphatase in human cells.

Fasina YO, Thanissery RR (2011). Comparative efficacy of a yeast product and bacitracin methylene disalicylate in enhancing early growth and intestinal maturation in broiler chicks from breeder hens of different ages. Poult Sci. 90(5):1067-73. Assay: Phosphatase in chicken intestine.

Lopez-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. Assay: Phosphatase in mouse cell supernatant.

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. Assay: Phosphatase in fish marine teleost follicles, eggs.

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

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. Assay: Phosphatase in chicken intestine.

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. Assay: Phosphatase in human cells.

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. Assay: Phosphatase in bacteria cells.

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. Assay: Phosphatase in chicken intestinal tissue.

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. Assay: Phosphatase in mouse cell supernatant.

Monick, M.M. et al (2006). Active ERK Contributes to Protein Translation by Preventing JNK-Dependent Inhibition of Protein Phosphatase. J. Immunol. 177: 1636-1645. Assay: Phosphatase in human cells.

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

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. Assay: Phosphatase in marine teleost oocyte.

To find more recent publications, please click here.

If you or your labs do not have the equipment or scientists necessary to run this assay, BioAssay Systems can perform the service for you.

– Fast turnaround
– Quality data
– Low cost