Alkaline Phosphatase Activity Fluorometric Assay Kit Kit
- Alkaline Phosphatase (ALP)
- Detection Method
- Minimum Detection Limit
- 1 μU
- Activity Assay (AcA)
- Sample Type
- Cell Lysate, Plasma, Serum, Tissue Samples
- In Alkaline Phosphatase Fluorimetric Assay Kit, ALP cleaves the phosphate group of the non-fluorescent 4- Methylumbelliferyl phosphate disodium salt (MUP) substrate resulting in an intense fluorescent signal (Ex/Em = 360nm/440nm). The kit is an ultra sensitive, simple, direct and HTS-ready assay designed to measure ALP activity in serum and bio- samples with detection sensitivity approx. 1 μU, more sensitive than colorimetric assays. The kit is suitable for both research and drug discovery.
- Alkaline Phosphatase Activity Fluorometric Assay Kit: High-Throughput Ready Fluorometric Assay to Measure Alkaline Phosphatase (ALP) activity in Cells, Serum, Plasma etc. High sensitivity, Simple & Direct.
ALP Assay Buffer
- Application Notes
- The assay designed to measure ALP activity in serum and bio-samples with detection sensitivity ~1 μU
Further details regarding sample type: Cell and tissue lysates, culture media, urine, plasma and serum, as well as many other biological fluids
1. Sample Preparations: Inhibitors of ALP, like tartrate, fluoride, EDTA, oxalate, and citrate, should be avoided in sample preparation. Serum, plasma, urine, semen, and cell culture media can be assayed directly. Cells (1×10 5 ) or tissue (approx. 10 mg) can be homogenized in 100 µL Assay Buffer, centrifuge to remove insoluble material at 13,000g for 3 minutes. Add test samples directly into 96-well plate, bring total volume to 110 µL with Assay Buffer. In order to avoid interference of components in the sample, set a sample background control. Add the same amount of samples into separate wells, bring volume to 110 µL. Add 20 µL Stop Solution and mix well to terminate ALP activity in the sample.
2. Dilute enough 5 mM MUP substrate solution to 0.5 mM with Assay Buffer (1:10), add 20 µL of the 0.5 mM MUP substrate solutions to each well containing the test samples and background controls. Mix well. Incubate the reaction for 30 min (or longer if ALP activity in sample is low) at 25 °C, protect from light.
3. Standard Curve: Dilute 10 µL of the 5 mM MUP solution with 990 µL Assay Buffer to generate 50 µM MUP standards. Add 0, 2, 4, 6, 8, 10 µL into 96-well plate in duplicate to generate 0, 0.1, 0.2, 0.3, 0.4, 0.5 nM/well MUP standard. Bring the final volume to 120 µL with Assay Buffer. Add 10 µL of ALP enzyme solution to each well containing the MUP standard. Mix well. Incubate the reaction for 30 min at 25 °C, protect from light. The ALP enzyme will convert MUP substrate to equal amount of fluorescent 4-Methylumbelliferone (4-MU).
4. Stop all reactions by adding 20 µL Stop Solution into each standard and sample reaction except the sample background control reaction (since 20 µL Stop Solution has been added into the background control when prepare the sample background control in step 1), gently shake the plate. Measure fluorescence intensity at Ex/Em 360/440 nm using a fluorescence microtiter plate reader.
- Calculation of Results
Calculation: Correct background by subtracting the value derived from the sample background controls for samples. Plot 4-MU standard Curve. Apply sample readings to the standard curve to get the amount of 4-MU generated by ALP sample. ALP activity of the test samples can be calculated: ALP activity= A/V/T (mU/mL) Where: A is amount of 4-MU generated by samples (in nmol). V is volume of sample added in the assay well (in ml). T is reaction time (in minutes). VI. Unit Definition : The amount of enzyme causing the hydrolysis of 1 µM of MUP per minute at pH 10.0 and 25 °C (glycine buffer).
- For Research Use only
- -20 °C
- Expiry Date
- 12 months
Holscher, Davis, Tappenden: "Human milk oligosaccharides influence maturation of human intestinal Caco-2Bbe and HT-29 cell lines." in: The Journal of nutrition, Vol. 144, Issue 5, pp. 586-91, 2014 (PubMed).
- Holscher, Davis, Tappenden: "Human milk oligosaccharides influence maturation of human intestinal Caco-2Bbe and HT-29 cell lines." in: The Journal of nutrition, Vol. 144, Issue 5, pp. 586-91, 2014 (PubMed).
- Alkaline Phosphatase (ALP)
- Alternative Name
- Alkaline Phosphatase
- ALP, ALP1, CRASH, ECK0378, JW0374, psiA, 2410004D18Rik, AU040643, AW060375, ILC, CTACK, ESkine, Akp2, alp, zgc:56672, DDBDRAFT_0205491, DDBDRAFT_0231570, DDB_0205491, DDB_0231570, Actn2lp, Alp, asparaginase like 1, bacterial alkaline phosphatase, C-C motif chemokine ligand 27, alkaline phosphatase, liver/bone/kidney, alkaline phosphatase, PDZ and LIM domain 3, ASRGL1, phoA, Asrgl1, CCL27, alpl, alp, Pdlim3
- Alkaline phosphatase (ALP) catalyzes the hydrolysis of phosphate esters in alkaline buffer and produces an organic radical and inorganic phosphate. The change in alkaline phosphatase level and activity is associated with a lot of diseases in the liver and bones. Alkaline phosphatase is also a popular enzyme conjugated to secondary antibody in ELISA. In