Alkaline Phosphatase Assay Kit (Colorimetric)

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1 ab83369 Alkaline Phosphatase Assay Kit (Colorimetric) Instructions for Use For the rapid, sensitive and accurate measurement of Alkaline Phosphatase in various samples This product is for research use only and is not intended for diagnostic use.

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3 Table of Contents 1. Overview 3 2. Protocol Summary 4 3. Components and Storage 5 4. Assay Protocol 7 5. Data Analysis 9 6. Troubleshooting 11 2

4 1. Overview 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. Abcam s Alkaline Phosphatase Assay Kit (Colorimetric) is a highly sensitive, simple, direct and HTS-ready colorimetric assay designed to measure ALP activity in serum and biological samples. It contains 10 substrate tablets providing convenience for multiple usages. The kit uses p-nitrophenyl phosphate (pnpp) as a phosphatase substrate which turns yellow (λ max = 405 nm) when dephosphorylated by ALP. The Kit can detect µu ALP in samples. 3

5 2. Protocol Summary Sample Preparation Add pnpp to Samples Standard Curve Preparation Incubate with ALP Enzyme Solution Add Stop Solution A Measure Optical Density 4

6 3. Components and Storage A. Kit Components Item Quantity ALP Assay Buffer 100 ml pnpp (10 tablets) 1 vial ALP Enzyme 1 vial Stop Solution 10 ml Store the kit at -20 C, protect from light. Allow Assay Buffer to warm to room temperature before use. Briefly centrifuge vials before opening. Keep samples, ALP Enzyme and pnpp solution on ice during the assay. Read the entire protocol before performing the assay. pnpp SOLUTION: Dissolve 2 tablets pnpp into 5.4 ml Assay Buffer to make 5 mm work solution. Two tablets are sufficient for 100 assays. NEVER TOUCH THE TABLETS WITH BARE HANDS. The pnpp solution is stable for 12 hours on ice. ALP ENZYME: Reconstitute ALP Enzyme with 1 ml Assay Buffer. DO NOT FREEZE! The enzymes are stable for up to 2 month at 4 C after reconstitution. 5

7 B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Colorimetric microplate reader 96 well plate Orbital shaker 6

8 4. Assay Protocol 1. Sample Preparation: Note: Inhibitors of ALP, such as EDTA, oxalate, fluoride, and citrate should be avoided in sample preparation. a. For serum and plasma samples: Serum and plasma should be diluted 10 times; cell culture media can be measured directly. b. For cell samples: To measure intracellular ALP, washed cells ( ) can be homogenized in the Assay Buffer, centrifuge to remove insoluble material at 13,000 x g for 3 minutes. Add different volume of samples into 96-well plate; bring the total volume to 80 µl with Assay Buffer. Note: Colored samples may interfere with OD 405nm 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. 7

9 3. Standard Curve Preparation: Dilute 40 µl of the 5 mm pnpp solution with 160 µl Assay Buffer to generate 1 mm pnpp standard. Add 0, 4, 8, 12, 16, 20 µl into 96-well plate in duplicate to generate 0, 4, 8, 12, 16, 20 nmol/well pnpp standard. Bring the final volume to 120 µl with Assay Buffer. 4. ALP Conversion: Add 10 µl of ALP enzyme solution to each well containing the pnpp standard. Mix well. The ALP enzyme will convert pnpp substrate to an equal amount of colored p-nitrophenol (pnp). Incubate the reaction for 60 min at 25 C, protect from light. 5. 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. 6. Measure OD at 405 nm in a microplate reader. 8

10 5. Data Analysis Correct background by subtracting the value derived from the zero standards from all standards, samples and sample background control The background reading can be significant and must be subtracted from sample readings. Plot pnp Standard Curve. Apply sample readings to the standard curve to get the amount of pnp 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 pnp generated by samples (in µmol). V is volume of sample added in the assay well (in ml). T is reaction time (in minutes) 9

11 Unit Definitions: All the Units mentioned in this protocol are Glycine Units. Glycine Units: The amount of enzyme causing the hydrolysis of one micromole of pnpp per minute at ph 9.6 and 25 C (glycine buffer). DEA Units: The amount of enzyme causing the hydrolysis of one micromole of pnpp perminute 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. 10

12 6. Troubleshooting Problem Reason Solution Assay not working Unexpected results Assay buffer at wrong temperature Protocol step missed Plate read at incorrect wavelength Unsuitable microtiter plate for assay Measured at wrong wavelength Samples contain impeding substances Unsuitable sample type Sample readings are outside linear range Assay buffer must not be chilled - needs to be at RT Re-read and follow the protocol exactly Ensure you are using appropriate reader and filter settings (refer to datasheet) Fluorescence: Black plates (clear bottoms); Luminescence: White plates; Colorimetry: Clear plates. If critical, datasheet will indicate whether to use flat- or U-shaped wells Use appropriate reader and filter settings described in datasheet Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet Concentrate/ dilute samples to be in linear range 11

13 Samples with inconsistent readings Lower/ Higher readings in samples and standards Unsuitable sample type Samples prepared in the wrong buffer Samples not deproteinized (if indicated on datasheet) Cell/ tissue samples not sufficiently homogenized Too many freezethaw cycles Samples contain impeding substances Samples are too old or incorrectly stored Not fully thawed kit components Out-of-date kit or incorrectly stored reagents Reagents sitting for extended periods on ice Incorrect incubation time/ temperature Incorrect amounts used Refer to datasheet for details about incompatible samples Use the assay buffer provided (or refer to datasheet for instructions) Use the 10kDa spin column (ab93349) Increase sonication time/ number of strokes with the Dounce homogenizer Aliquot samples to reduce the number of freeze-thaw cycles Troubleshoot and also consider deproteinizing samples Use freshly made samples and store at recommended temperature until use Wait for components to thaw completely and gently mix prior use Always check expiry date and store kit components as recommended on the datasheet Try to prepare a fresh reaction mix prior to each use Refer to datasheet for recommended incubation time and/ or temperature Check pipette is calibrated correctly (always use smallest volume pipette that can pipette entire volume) 12

14 Problem Reason Solution Standard curve is not linear Not fully thawed kit components Pipetting errors when setting up the standard curve Incorrect pipetting when preparing the reaction mix Air bubbles in wells Concentration of standard stock incorrect Errors in standard curve calculations Use of other reagents than those provided with the kit Wait for components to thaw completely and gently mix prior use Try not to pipette too small volumes Always prepare a master mix Air bubbles will interfere with readings; try to avoid producing air bubbles and always remove bubbles prior to reading plates Recheck datasheet for recommended concentrations of standard stocks Refer to datasheet and re-check the calculations Use fresh components from the same kit For further technical questions please do not hesitate to contact us by or phone (select contact us on for the phone number for your region). 13

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16 UK, EU and ROW Tel: +44 (0) US, Canada and Latin America Tel: ABCAM (22226) China and Asia Pacific Tel: ( 中國聯通 ) Japan Tel: +81-(0) Copyright 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print.

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