ATP Luminometric Assay Kit

Transcription

1 ATP Luminometric Assay Kit Catalog No. KM0029 Detection and Quantification of ATP Concentrations in Biological Samples. Research Purposes Only. Not Intended for Diagnostic or Clinical Procedures.

2 INTRODUCTION Adenosine triphosphate (ATP) is a major metabolite in cellular energetics and signaling, therefore functioning in the regulation of cell metabolism and other processes. It is an energy source for virtually all living organisms that rapidly degrades in absence of viable organisms. Thus, its existence can be used to identify and verify the presence of viable organisms. ATP quantification has many biological applications, including cell cytotoxicity, detection of bacteria on surfaces, quantifying bacteria in water and somatic cells in culture, and analyzing food quality. This ultrasensitive assay allows for determination of cell proliferation and cytotoxicity in mammalian cells. ATP has been found to be essential in light production in bioluminescence of firefly luciferase, a monomeric 61 kd enzyme that catalyzes a two-step oxidation of luciferin to yield light at 560 nm. The first step consists of protein activation by ATP to produce an intermediate that is a reactive mixed anhydride. This intermediate reacts with oxygen in a second step to create a transient dioxetane, which is quickly broken down into the oxidized products of oxyluciferin and carbon dioxide, along with the burst of light. With ATP as a limiting component, this light intensity is proportional to ATP concentration and can be monitored at 560 nm with a luminometer or other luminescence instruments. FEATURES Number of Assays: 100 Assays Luminometric Dynamic Range: 1 to 1000 nm ATP Luminescence at 560 nm STORAGE & HANDLING Upon receipt, store all other 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 1 plate RT Reaction Buffer 10 ml -20 C Reaction Enzyme 1 vial -20 C Dye Reagent 1 vial -20 C Note: If not running entire plate at once, aliquot and store reagents under conditions according to this table. Use within 2 months. MATERIALS NOT PROVIDED ATP Standard, Pipetting devices, tubes, and microplate reader. 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 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 Cells (or samples) For adherent cells: Plate cells overnight in growth medium at 1,000-10,000 cells/90 ul/well. For non-adherent cells: Centrifuge the cells from the culture medium and then suspend the cell pellets in culture medium at 2,000-20,000 cells/90 ul/well for a 96-well poly-d lysine. Centrifuge the plate at 800 rpm for 2 minutes with brake off prior to the experiments. Note1: Each cell line should be evaluated on an individual basis to determine the optimal cell density for proliferation or cytotoxicity induction. For toxicity assays, start with more cells. Note2: For all luminescent experiments, it is recommended to use white plates to achieve the best results. 2. Preparation of Reaction Mix Thaw all components to room temperature before use. Add 10 ml of Reaction Buffer into a Dye Reagent bottle and mix well. Then add 20ul of Reaction Enzyme into reconstituted Dye Reagent to create working Reaction Mix, and mix well. Note: Aliquot and store the unused components at -20 C, and avoid repeated freeze/thaw cycles and potential ATP contamination from exogenous biological sources. 3. Test Compounds Treat cells (or samples) with test compounds by adding 10 ul of 10X compounds for a 96-well plate in desired compound buffer. For blank wells (medium without the cells), add the corresponding amount of compound buffer. Incubate the cell plate in a 37 C, 5% CO 2 incubator for the desired period of time, such as 24, 48, or 96 hours. 4. Preparation of Standard (optional) To prepare the standard, dilute the appropriate amount of ATP stock solution in PBS with 0.1% BSA, starting from a high point of 1 µm down to a low point of 1 nm to create 7 concentrations for the standard. The last row consists of only buffer to serve as a negative control. The standards are run in duplicates. 3

5 ASSAY PROTOCOL Note: An ATP standard curve should be generated together with the above assay if the absolute amount of ATP in samples needs to be calculated. 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=ATP Standards, EX=Experimental Test Samples 1. For an ATP standard curve, add ATP standards into an empty lane with the last row being the control for background luminescence. The standards should be run in duplicates. 2. Add 100 ul of the freshly prepared working Reaction Mix to each well of the microplate containing the standards, controls, and samples. 3. Incubate at room temperature for 10 to 20 minutes, protected from light. 4. Read the luminescence intensity with a standard luminometer. 5. Generate ATP standard curve is needed. 4

6 STANDARD CURVE Correct for background absorbance. For each point, subtract the value derived from the negative control. Plot the ATP concentrations vs. the read values to create the standard curve. ATP Luminometric Assay Kit allows for the detection and quantification of endogenous ATP concentrations within the range of nm 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