SEAP Reporter Gene and Alkaline Phosphatase Activity Assay Kit (Luminometric)

Product details

Abcam's Luminometric SEAP Reporter Gene and Alkaline Phosphatase Activity Assay Kit (ab323460) utilizes a novel water-soluble chemiluminescent ALP substrate, to enable ultra-sensitive detection of ALP activity in biological fluids, whole cells and cell/tissue lysates as well as secreted alkaline phosphatase (SEAP) in culture medium from transfected cells. The assay can detect ALP and SEAP in the microunit/well range and can also be used to identify pluripotent/multipotent stem cells without requiring lysis or fixation.
Enzymatic hydrolysis of the phosphate ester moiety in the luminogenic substrate yields an intermediate product that undergoes rapid self-immolative chemiexcitation, emitting bright green light (λem = 510 nm). The luminescent substrate has a high quantum yield in aqueous solutions (up to 9.8%, compared with 0.03% for CDP-Star) and does not require any surfactants, enhancers or organic solvents to achieve maximum light emission. The luminescent probe is non-cytotoxic, so the kit can also be used to identify pluripotent or multipotent stem cells.
SEAP Reporter and ALP Activity Assay Protocol:
-Add samples to a white 96-well assay plate.
-If measuring SEAP excreted in culture medium, heat-inactivate endogenous serum alkaline photosphatase (from FBS) at 65°C.
-Make ALP Luminometric substrate master mix.
-Measure luminescence intensity in kinetic mode, reading every 1-2 minutes for at least 30 minutes. Peak luminescence is typically achieved within 5-10 min and signal intensity begins to decay after 15-30 minutes, depending upon the sample ALP activity.""
Most sensitive assay for alkaline phosphatase with 4-5 decades of dynamic range.