The recommended sample volume is 10 microliters. The higher concentration of 5 mM that was mentioned corresponds to 50 nanomoles. This would be near the upper limit of the assay.
Bioluminescent ATP assays are very sensitive and accurate over a broad range of the amount of ATP; nanomole and even picomole amounts of ATP can be determined. So, a 5 millimolar sample could be diluted 1000X to 5 micromolar and still give good results, and likely better results than the 5 mM sample.
MAK135
ADP/ATP Ratio Assay Kit
sufficient for 100 tests (bioluminescent)
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About This Item
UNSPSC Code:
12161503
NACRES:
NA.84
Pricing and availability is not currently available.
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usage
sufficient for 100 tests (bioluminescent)
application(s)
pharmaceutical
detection method
chemiluminescent
relevant disease(s)
cancer
storage temp.
−20°C
General description
Changes in the ADP/ATP ratio have been used to differentiate modes of cell death and viability. Increased levels of ATP and decreased levels of ADP signify proliferating cells. Conversely, decreased levels of ATP and increased levels of ADP represent apoptotic or necrotic cells where the decrease in ATP and increase in ADP are much more pronounced in necrosis versus apoptosis.
Application
- LOC554202 contributes to chordoma progression by sponging miR-377-3p and up-regulating SMAD3.: This article investigates the molecular mechanisms by which LOC554202 facilitates chordoma progression through miR-377-3p and SMAD3 regulation. The ADP/ATP Ratio Assay Kit was utilized to assess the metabolic impact of these molecular changes on cell viability and proliferation (Xu et al., 2023).
- FOXG1 improves mitochondrial function and promotes the progression of nasopharyngeal carcinoma.: This study explores how FOXG1 enhances mitochondrial function, thereby promoting nasopharyngeal carcinoma progression. The ADP/ATP Ratio Assay Kit was employed to measure mitochondrial function and energy metabolism changes, providing insights into the bioenergetic profile of cancer cells (Xi et al., 2021).
Features and Benefits
Compatible with high-throughput handling systems.
Suitability
Suitable for the detection of apoptosis and necrosis in cells and for the studying the effects of compounds on cellular proliferation.
Principle
The ADP/ATP Ratio Assay kit provides a simple and direct procedure for measuring ADP and ATP levels in cells for the screening of apoptosis, necrosis, and cell proliferation. The assay involves two steps. In the first step, the working reagent lyses cells to release ATP and ADP. In the presence of luciferase, ATP immediately reacts with the Substrate D-luciferin to produce light. The light intensity is a direct measure of the intracellular ATP concentration.
Luciferase
ATP + D-Luciferin + O2 ----------> oxyluciferin + AMP + PPi + CO2 + light
In the second step, the ADP is converted to ATP through an enzyme reaction. This newly formed ATP then reacts with the D-luciferin as in the first step. The second light intensity measured represents the total ADP and ATP concentration in the sample.
Luciferase
ATP + D-Luciferin + O2 ----------> oxyluciferin + AMP + PPi + CO2 + light
In the second step, the ADP is converted to ATP through an enzyme reaction. This newly formed ATP then reacts with the D-luciferin as in the first step. The second light intensity measured represents the total ADP and ATP concentration in the sample.
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Hazard Classifications
Aquatic Chronic 3 - Skin Sens. 1
Storage Class Code
10 - Combustible liquids
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What is the appropriate concentration range for the assay? Can I use it to test the ADP/ATP ratio in buffer (~ 0.5 - 5 mM ATP and ADP)?
1 answer-
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Can I please see the product insert and instruction for use for product codeMAK135 ADP/ATP Ratio Assay kit
1 answer-
Here is a link to the product protocol. It can also be found in the 'Documentation' section of the product page: https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/135/374/mak135bul.pdf
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Can the cells be stored in a freezer for future use with the MAK135-1KT ADP/ATP Ratio Assay Kit? Should the same approach recommended for tissue testing, involving snap freezing the tissue in liquid nitrogen and deproteinizing it to inactivate residual ATPases, be followed for testing cells?
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The approach for cells is similar to that for tissue samples, but with some differences. It is suggested to liquid nitrogen snap freeze the cells in DMSO to prevent cell lysis. Since these are cells, a deproteination step is not necessary. After thawing, the cells should be washed to remove the DMSO and replaced with PBS. Adding a phosphatase inhibitor such as sodium orthovanadate to the PBS can help limit undesired ATPase activity. It is advisable to resuspend the cells in an amount of PBS that allows them to dispense 10^3-10^4 cells in 10 uL sample volumes, as per the ELDT protocol. Quick work is advised, especially after thawing, to initiate the assay, as snap freezing and thawing the cells can stress them. It's preferable to assay freshly harvested cells over snap freezing, as the kit has only been tested on fresh cells.
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How should this item be used on tissue samples? What is the preparation process and what type of buffer should be used?
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Given the relatively unstable nature of ATP, if immediate processing of the tissue and running the assay is not feasible after harvesting the tissue, it is advisable to snap freeze the tissue in liquid nitrogen. Prior to utilizing the tissue in the assay, it is recommended to deproteinate the tissue to deactivate any residual ATPases. This process involves initially homogenizing the tissue on ice in PBS + 1 mM EDTA + 0.5% Triton X100, deproteinating samples with TCA, then neutralizing to pH 7 with KOH. Following this, the sample should be centrifuged and the clear supernatant utilized for the assay. An alternative method involves using a 10 kDa spin column for the lysate.
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