Alkaline Phosphatase Fluorimetric Assay Kit

Transcription

1 Alkaline Phosphatase Fluorimetric Assay Kit Catalog No. KM0019 Detection and Quantification of Alkaline Phosphatase Concentrations in Biological Samples. Research Purposes Only. Not Intended for Diagnostic or Clinical Procedures.

2 INTRODUCTION Alkaline Phosphatase (ALP) is a hydrolase enzyme that is most effective in alkaline environments and removes phosphate groups from various molecules, dephosphorylating alkaloids, nucleotides, and proteins. One common application of ALP is as a label for enzyme immunoassays. It is highly sensitive for ELISA-based diagnosis as well as biological assays like immunohistochemistry as well as Northern, Southern, and Western blots. Abnormal levels of ALP have clinical and diagnostic significance. For example, high levels of ALP in the body may indicate bile duct blockage, bone formation, or abnormalities related to the kidney or liver. This mix-and-read assay quantifies ALP activity in solutions, cell extracts, and solid surfaces with FDP. It uses a fluorogenic phosphatase substrate, which yields a fluorescent product that can be read with a fluorescence microplate reader with excitation at 490 nm and emission at 525 nm. FEATURES Number of Assays: 500 Assays Fluorometric Dynamic Range: 0.1 to 100 mu/ml Alkaline Phosphatase Fluorescence at Ex/Em = 490/525 nm STORAGE & HANDLING Upon receipt, store all components in the original box at -20 C, protected from light. The kit should be stable for at least six months. Note: Reagents provided in this kit may be harmful if ingested, inhaled or absorbed through the skin. 1

3 KIT CONTENTS & STOCK PREPARATION Reagent Amount Storage 96-Well Microplate 2 plates RT Reaction Buffer 25 ml -20 C Alkaline Phosphatase Standard 1 Vial of 10 U -20 C DMSO 500 µl -20 C Dye Reagent (light sensitive) 1 Vial -20 C Stop Solution 25 ml -20 C Note: If not running 500 assays at once, aliquot and store reagents at -20 C. Use within 2 months. MATERIALS NOT PROVIDED Pipetting devices, tubes, and microplate reader, black 96-well microplates, BSA ASSAY RESTRICTIONS This kit is intended for research purposes only, NOT diagnostic or clinical procedures of any kind. Materials included in this kit should NOT be used past the expiration date on the kit label. Reagents or substrates included in this kit should NOT be mixed or substituted with reagents or substrates from any other kits. Variations in pipetting technique, operator laboratory technique, kit age, incubation time or temperature may cause differences in performance of the materials provided. The assay is designed to eliminate interference and background by other cellular macromolecules or factors present within any biological samples. However, the possibility of background noise cannot be fully excluded until all factors have been tested using the assay kit. 2

4 REAGENT & SAMPLE PREPARATIONS FOR 1 PLATE Please read the manual in its entirety before proceeding with the assay. Note: Warm all reagents to room temperature before use and protect from light. Aliquot and store the reagents at -20 C if not all of it is used. 1. Preparation of Standard Add 100 µl of distilled water with 0.1% BSA to the Alkaline Phosphatase Standard to make a 100 U/ml stock concentration. To prepare the standard, dilute the appropriate amount of Alkaline Phosphatase stock solution in distilled H 2 O with 0.1% BSA, starting from a high point of 100 mu/ml down to a low point of 0.1 mu/ml to create 7 concentrations for the standard. The last row consists of only distilled H 2 O with 0.1% BSA to serve as a negative control. The standards are run in duplicate. Use a 96-well black, flat bottom plate. Note: Unused serial diluted alkaline phosphatase standard should be discarded. 2. Preparation of Samples Dilute the samples in distilled H 2 O (with 0.1% BSA if needed) or as desired. A variable dilution will be required depending on the total amount of Alkaline Phosphatase present in your sample. Note: If using cell or tissue samples, prepare them as desired. 3. Preparation of Dye Reagent Prepare the Dye Reagent stock solution by adding 100 µl of DMSO into a vial of Dye Reagent. Use promptly. If not all of it is used, aliquot and store at -20 C. Avoid multiple freeze and thaw cycles. 4. Preparation of Working Solution Add 20 µl of the prepared Dye Reagent into 5 ml of Reaction Buffer to make the Working Solution. Mix well by inversion. Avoid exposure to light. 3

5 PROTOCOL FOR ASSAY IN SUPERNATANTS (1 PLATE) 1. Add 50 µl of each sample/standard to each well in a 96-well flat bottom microplate. The standards are run in duplicate. It is advised to run the samples in duplicate. Sample Plate Layout (you do not have to follow this) A ST1 ST1 EX EX B ST2 ST2 EX C ST3 ST3 D ST4 ST4 E ST5 ST5 F ST6 ST6 G ST7 ST7 H BL BL Note: BL=Blank Control, ST=Alkaline Phosphatase Standards, EX=Experimental Test Samples 2. Add 50 µl of the Working Solution to each well of the microplate containing the standards, controls, and samples. 3. Incubate at room temperature for 10 to 30 minutes, protected from light. 4. Optional: Add 50 µl of Stop Solution to each well of the microplate containing the standards, controls, and samples. 5. Read the plate using a fluorescence microplate reader at Ex/Em: 490/525 nm. PROTOCOL FOR ASSAY IN CELLS (1 PLATE) 1. Treat cells as desired. 2. Remove the growth medium from the cell plate completely. The growth medium may interfere with the Dye Reagent. 3. Make a 1:1 dilution of 5 ml of Working Solution with 5 ml of distilled H2O. 4. Add 100 µl of 1:1 diluted Working Solution into wells with cells. 5. Incubate at desired temperature for 30 to 60 minutes, protected from light. 6. Optional: Add 50 µl of Stop Solution to each well of the microplate containing the standards, controls, and samples. 7. Read the plate using a fluorescence microplate reader at Ex/Em: 490/525 nm. 4

6 STANDARD CURVE Correct for background fluorescence or absorbance. For each point, subtract the negative control from the value derived. Plot the Alkaline Phosphatase concentrations vs. the read values to create the standard curve. Alkaline Phosphatase Fluorometric Assay Kit allows for the detection and quantification of endogenous Alkaline Phosphatase concentrations within the range of 0.1mU/ml - 100mU/ml in cell lysates, sera, and plasma. 5

7 TECHNICAL SUPPORT For troubleshooting, information or assistance, please go online to or contact us at NOTES 6