Alkaline Phosphatase Activity Colorimetric Assay Kit Kit
- Alkaline Phosphatase (ALP)
- Detection Method
- Minimum Detection Limit
- 10-250 μU
- Activity Assay (AcA)
- Sample Type
- Cell Lysate, Plasma, Serum, Tissue Samples
- Assay Kit is a high sensitivity, simple, direct and HTS-ready colorimetric assay designed to measure AP activity in serum and other samples. It is suitable for research and drug discovery. The kit uses p -nitrophenyl phosphate (p NPP) as a phosphatase substrate which turns yellow (lambda max = 405 nm) when dephosphorylated by AP. The kit can detect µU acid phosphatase activity in samples.
- Alkaline Phosphatase Activity Colorimetric Assay Kit: High-Throughput Ready Colorimetric Assay to Measure Alkaline Phosphatase (ALP) activity in Cells, Serum, Plasma etc. High sensitivity, Simple & Direct.
ALP Assay Buffer
pNPP (10 TAB)
- Application Notes
- The Kit can detect 10-250 μU ALP in samples.
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, such as EDTA, oxalate, fluoride, and citrate should be avoided in sample preparation. Serum and plasma shoule be diluted 10 times, cell culture media can be measured directly. To measure intracellular ALP, washed cells (1x10^5 ) can be homogenized in the Assay Buffer, centrifuge to remove insoluble material at 13,000g for 3 minutes. Add different volume of samples into 96-well plate, bring the total volume to 80 µL with Assay Buffer. Colored samples may interfere with O.D. 405 nm readings, so use a sample background control. Add the same amount of sample into separate wells, bring volume to 80 µL. Add 20 µL stop solution and mix well to terminate ALP activity in the sample.
2. Add 50 µL of the 5 mM pNPP solution to each well containing the test samples and background controls. Mix well. Incubate the reaction for 60 min at 25 °C, protect from light.
3. Standard Curve: Dilute 40 µL of the 5 mM p NPP solution with 160 µL Assay Buffer to generate 1 mM p NPP standard. Add 0, 4, 8, 12, 16, 20 µL into 96-well plate in duplicate to generate 0, 4, 8, 12, 16, 20 nM/well p NPP standard. Bring the final volume to 120 µL with Assay Buffer. Add 10 µL of ALP enzyme solution to each well containing the p NPP standard. Mix well. The ALP enzyme will convert p NPP substrate to an equal amount of colored p -Nitrophenol (p NP). Incubate the reaction for 60 min at 25 °C, protect from light.
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 to the background control when prepared in step 1), gently shake the plate. Measure O.D. at 405 nm in a micro plate reader.
- Calculation of Results
Calculation: Correct background by subtracting the value derived from the 0 standards from all standards, samples and sample background control (The background reading can be significant and must be subtracted from sample readings). Plot p NP Standard Curve. Apply sample readings to the standard curve to get the amount of p NP generated by ALP sample. ALP activity of the test samples can then be calculated: ALP activity(U/mL) = A/V/T Where A is amount of p NP generated by samples (in µM). V is volume of sample added in the assay well (in ml). T is reaction time (in minutes) VI. Unit Definition: All the Units mentioned in this protocol are Glycine Units. Glycine Units: The amount of enzyme causing the hydrolysis of one micromole of p NPP per minute at pH 9.6 and 25 °C (glycine buffer). DEA Units: The amount of enzyme causing the hydrolysis of one micromole of p NPP per minute at pH 9.8 and 37 °C (diethanolamine buffer). Unit Conversion: One Glycine unit as described above is equivalent to approximately three DEA units. This reaction system is in Glycine buffer. Note: Ensure that the Assay Buffer is at room temperature before use. Keep samples, ALP Enzyme and pNPP solution on ice during the assay. V. Alkaline Phosphatase
- For Research Use only
- -20 °C
- Expiry Date
- 12 months
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- 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. Changes in alkaline phosphatase level and activity are associated with various disease states in the liver and bone.