MFP 蛋白磷酸酶检测试剂盒 荧光法、MFP Protein Phosphatase Assay Kit *Fluorimetric*详细如下: 蛋白磷酸酶作为药物筛选靶点受到了极大的关注。MFP 是大多数磷酸酶(即碱性磷酸酶、酸性磷酸酶、蛋白酪氨酸磷酸酶和丝氨酸/苏氨酸磷酸酶)的专有荧光底物。磷酸酶诱导的 MFP 水解产生强烈的荧光产物,其激发波长约为 470 nm,最大发射波长约为 510 nm。这一特性使基板适用于所有荧光仪器。MFP 蛋白磷酸酶检测试剂盒使用 MFP 量化蛋白磷酸酶活性。该试剂盒可用于表征酶反应动力学和蛋白磷酸酶抑制剂的高通量筛选。检测限:5 ng/ml Start the protein phosphatase detection. Add 50 uL/well of the protein phosphatase-containing sample. Include a nonphosphatase-containing sample as a negative control. Add 50 uL/well of the MFP reaction solution. Mix the reagents by gently shaking the plate for 30 sec. Measure fluorescence signal: • For kinetic reading: Immediately start measuring fluorescence intensity at Ex/Em=470±20 nm/510±20 nm continuously and record data every 5 min for 30 to 60 min. • For end-point reading: Incubate the reaction at the desired temperature for 30 to 60 min, and keep away from light. Optional: Add 50 uL/well of stop solution (Component E). Measure fluorescence intensity at Ex/Em=470±20 nm/510± 20 nm. • Prepare 1X lysis buffer by adding 1 mL of 10X lysis buffer (Component C) to 9 mL of deionized water. • Wash cells with 1X lysis buffer twice gently. • Add 20uL of Triton X-100 (Component D) to 10 mL of 1X lysis buffer. Add protease inhibitors to a final concentration of 10ug/ml Aprotinin, 10ug/mL Leupeptin, 100uM PMSF and 10ug/ml Pepstatin A. Note: Protease inhibitors are not provided. • Add an appropriate amount of the above lysis buffer to the cells or cell pellet. Scrape off the adherent cells or resuspend the cell pellet. Collect the cell suspension in a microcentrifuge tube. • Incubate the cell suspension at 4℃ for 10 min with agitation. • Centrifuge the cell suspension at 2500 X g for 10 min at 4℃. • Collect the supernatant to perform the protein phosphatase assay. Prepare 1X lysis buffer by adding 1 mL of 10X lysis buffer (Component C) and 20 uL of Triton X-100 (Component D) to 9 mL of deionized water. Add protease inhibitors to a final concentration of 10ug/ml Aprotinin, 10ug/mL Leupeptin, 100uM PMSF, and 10ug/ml Pepstatin A. Note: Protease inhibitors are not provided. • Add an appropriate amount of 1X lysis buffer to the tissue sample, and homogenize. • Centrifuge the tissue sample at 10,000 X g for 10 min at 4℃. • Collect the supernatant for protein phosphatase assay.