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AnaSpec Introduces Line of Assay Kits for Phosphatase Detection
Protein phosphatases play key cellular regulatory roles in such processes as differentiation, proliferation and apoptosis (1). As a result, they have received significant attention as potential drug-screening targets. AnaSpec’s range of protein phosphatase assay kits include both fluorimetric and colorimetric readouts. The colorimetric assay kit utilizes p-nitrophenyl phosphate (pNPP) as a substrate. It shows high sensitivity and a wide linear range. The detection limit is generally 3 ng or lower. The fluorimetric assays are even more sensitive than the colorimetic assay, and there are two kits to choose from. The EnzoLyteTM FDP Assay Kit, using FDP (3,6-Fluorescein Diphosphate), is optimized for assaying protein phosphatases which require pH>6.5 for optimal activity, while the EnzoLyteTM MFP Assay Kit is for phosphatases which require pH< 6.5. Both fluorophores emit at about 520 nm upon dephosphorylation.
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The change in alkaline phosphatase level and activity is involved in a variety of physiological and pathological events, such as bone development (2), bone-related diseases (3), gestation related diseases (4), inflammatory bowel disease (5), post-parathyroidectomy stage (6), and drug toxicity (7). Alkaline phosphatase is also widely used, when conjugated to a secondary antibody, as a reporter in ELISA assays. The Enzolyte Alkaline Phosphatase Assay Kits are designed to detect enzyme activity in biological samples, while the EnzoLyteTM Alkaline Phosphatase ELISA Assay Kits are optimized to detect alkaline phosphatase in ELISA.
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The placental alkaline phosphatase is the most stable isoenzyme among the four mammalian alkaline phosphatase isoenzymes, and it only exists naturally in the placenta of higher primates. These characteristics make placental alkaline phosphatase the alkaline phosphatase of choice to serve as a reporter gene for the analysis of promoter activity and gene expression in cell culture or animals. The natural form of placental alkaline phosphates is membrane-anchored. The recombinant form of placental alkaline phosphatase, secreted alkaline phosphatase (SEAP), can be efficiently secreted into tissue culture medium and serum (8, 9). This unique characteristic of SEAP provides the capability of performing continuous kinetic analysis of gene expression over a period of time using a single cell culture or animal.
The EnzoLyteTM FDP Secreted Alkaline Phosphatase Reporter Gene Assay Kits assay both secreted and membrane-bound placental alkaline phosphatase. The fluorimetric assay kit uses FDP as the fluorogenic phosphatase substrate. Fluorescein, the final hydrolytic product of FDP, has a very high extinction coefficient and emission quantum yield. As a result, the assay is more sensitive than the colorimetric kit, which uses pNPP. Using FDP can detect as low as 0.1 ng of phosphatase with a linear range of three orders of magnitude, while the limit of detection using pNPP is 10 ng.
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The change in acid phosphatase level and activity is involved in a variety of physiological and pathological events, such as prostate puberty, rheumatoid arthritis (10), bone-resorption related diseases (11), and diabetes (12). Acid phosphatase is also a serum marker of tumor bone metastasis (13, 14).
The EnzoLyteTM MFP Acid Phosphatase Assay Kit is optimized to measure acid phosphatase activities using MFP as a fluorogenic substrate.
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The EnzoLyteTM MG Phosphate Assay Kit is based on the quantification of the blue-green complex formed between malachite green (MG) molybdate and free phosphate. This non-radioactive colorimetric assay kit has been optimized to offer superior sensitivity with a detection limit of as low as 8 pmoles of phosphate and an extended linear range of 0.1-50 μM phosphate. The assay has been widely used to quantify liberated phosphate in phosphatase assays, lipase assays, and nucleotide triphosphatase assays.
For more information visit www.anaspec.com
References:
1. Kupcho, K. et al. J. Biomol. Screening. 9, 223 (2004).
2. Kotobuki, N. et al. Cell Transplant. 13, 377 (2004).
3. Wyckoff, MH. et al., J. Clin. Endocrinol. Metab. 90, 1233 (2005).
4. Boronkai, A. et al., J.Clin.Pathol. 58, 72-76 (2005).
5. Sanchez, DM. et al., Biochem. Pharmacol. 68, 2317 (2004).
6. Morrone, LF. et al. Ann. Ital. Med. Int. 19, 189 (2004).
7. Papaldo, P. et al., Cancer Invest 22, 650 (2004).
8. Berger, J. et al. Gene 66, 1 (1988).
9. Cullen, BR. and MH. Malim, Methods Enzymol. 216, 362 (1992).
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