硝基苯酸钠（pNPP）磷酸酶检测试剂盒

产品：硝基苯酸钠（pNPP）磷酸酶检测试剂盒
货号：8108

Introduction

Protein phosphatase, enzyme that control the removal of phosphate （PO4^3-） group from protein molecules, regulates many fundamental cellular process such as cell attachment, proliferation, differentiation ａnd apoptosis. ScienCellT pNPP Phosphatase Assay is optimized to detect phosphatase activity in biological samples using pNPP （4-nitrophenyl phosphate） as a colorimetric substrate for most phosphatases. A water soluble yellow product with a strong absorption at 405 nm is developed during the reaction of pNPP with phosphatase ａnd can be detected with an ELISA plate reader.

Kit Components

Quality Control

Phosphatase, acid from potato （Sigma cat. P1146） ａnd alkaline from bovine intestinal mucosa （Sigma cat. P6772） were serially diluted in Assay Buffer-acidic ａnd Assay Buffer-alkaline respectively. The activity of both phosphatases were measured with ScienCellT pNPP Phosphatase Assay after a given time of reaction （15 min ａnd 45 min）, as shown in Figure 1 ａnd 2.

Procedures （96-well plate）

1. Preparation of phosphatase sample
   1. Prepare serial dilutions of phosphatase samples using the appropriate Assay Buffer with compatible pH （i.e.: Assay Buffer-acidic for acidic phosphatases, Assay Buffer-neutral for most neutral phosphatases, ａnd Assay Buffer-alkaline for alkaline phosphatases）.
   2. We recommend a concentration range of 0-100 µg/ml for the phosphatase dilutions. Phosphatases with very high or low activity may require use of lower or higher concentrations to ensure that the absorbance reading is in the linear range.
   3. A standard curve of phosphatase with known activity can be established.
2. Assay procedure
   1. Apply 45 µl of each phosphate solution ａnd 5 µl of 10× pNPP stock t to each well of the 96-well plate. Mix well ａnd incubate for the desired period of time （10-60 minutes） at 37ºC.
   2. Stop the reaction by adding 50 µl of Stop Buffer to each well, mix well ａnd measure the absorbance on an ELISA plate reader with a test wavelength at 405 nm ａnd a reference wavelength at 630 nm, ａnd subtract the 630 nm background absorbance from the 405 nm measurement.