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Product Details
| Cat # +Size | K354-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Absorbance (570 nm) or Fluorescence (Ex/Em 535/587 nm) |
| Species Reactivity | All |
| Applications | The assay can detect as low as 50 picomol (1 µM) of ATP in various samples. |
| Features & Benefits | • Simple procedure; takes less than 1 hour • Fast and convenient • Kit is designed to be a robust method which utilizes the phosphorylation of glycerol to generate a product that is easily quantified. |
| Kit Components | • ATP Assay Buffer • ATP Probe (in DMSO) • ATP Converter • Developer Mix (lyophilized) • ATP Standard (1 µmol; lyophilized) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
ATP is the primary energy currency of living systems. Virtually all energy requiring processes utilize the chemical energy stored in the phosphate bond of ATP. ATP is formed exclusively in the mitochondria and a variety of genetic diseases can affect ATP formation in the mitochondria. There are a number of commercially available ATP assays which detects femtomoles or less of ATP by measuring luminescence (BioVision Kit 254-200, for example) but these kits require specialized luminescence instrumentation and utilize luciferase which can be difficult to maintain in active form. BioVision newly developed ATP Colorimetric and Fluorometric Assay kit is designed to be a robust, simple method which utilizes the phosphorylation of glycerol to generate a product that is easily quantified by colorimetric (λmax = 570 nm) or fluorometric (Ex/Em = 535/587 nm) methods. The assay can detect as low as 50 picomol (1 µM) of ATP in various samples. The kit provides sufficient reagents for 100 assays.
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Shouldn't perchloric acid be added to the sample after homogenization with assay buffer, not before homogenization?
No, if you have access to the PCA, we recommend that you homogenize the samples in PCA. If not, you can use the provided assay buffer.
Do you have any recommendations for the type of 96 well plate to use for the fluorometric assay?
For the colorimetric assay, please use a clear plate. For the fluorometric assay a clear or a white plate can be used. Use flat bottom plates.
I have been trying to troubleshoot this customer. He is using human platelets for this kit (Ref: K354-100 - ATP Assay Kit) but the signal is extremely low. I have checked with him and the standard curve is fine so the kit seems to be working fine. Though the sample give very low signal. I have recommended increasing the cell numbers and using HeLa cells as good positive control which have high level of ATP. Do you have any suggestions for human platelets?
We have not used human platelets with this. Your suggestion of increasing the cell number sounds good. Also, have they checked for the quality of their platelets? In my experience it is tricky to get a pure fraction of healthy platelets and if there are a lot of lysed platelets in their samples, it would explain the low signals.
Could you please provide protocol instructions for the sample homogenization using perchloric acid and the following neutralisation?
Here is a general protocol:
1. Protein Precipitation: For biological samples with protein concentration less than ~ 20 mg/ml (tissue homogenate, cell lysate, urine, etc.), take 500 µl of sample, mix with 100 µl of ice cold PCA in 1.5 ml microfuge tubes, vortex briefly to mix well, place on ice for 5 min. Centrifuge at 13,000 x g for 2 min. Accurately transfer 480 µl of the supernatant to a fresh tube. For serum and other very high protein concentration samples, take 400 µl of sample and mix with 100 µl of ice-cold PCA, place on ice for 5 min. Centrifuge at 13,000 x g for 2 min. Accurately transfer 380 µl of the supernatant to a fresh tube. Depending on the nature of the analyte, the samples in PCA may be frozen at -70°C for up to a month for storage at this stage.
2. Sample Neutralization:Add 20 µl of ice-cold Neutralization Solution (resuspend the fine precipitate) and mix to neutralize the sample and precipitate excess PCA. There may be some gas (CO2) evolution so vent the sample tube. Place on ice for 5 min. Spin briefly (~ 1 - 2 min). Samples are now deproteinized, neutralized, and PCA has been removed. The samples may now be used in a variety of assays directly.As you can see, the general ratio for 1 N PCA vs 6 N KOH is 5:1. But, I would recommend a pH check after neutralization to see if it is within the range of 6.5 to 8.0. If not, you might need to add more KOH.
1. Protein Precipitation: For biological samples with protein concentration less than ~ 20 mg/ml (tissue homogenate, cell lysate, urine, etc.), take 500 µl of sample, mix with 100 µl of ice cold PCA in 1.5 ml microfuge tubes, vortex briefly to mix well, place on ice for 5 min. Centrifuge at 13,000 x g for 2 min. Accurately transfer 480 µl of the supernatant to a fresh tube. For serum and other very high protein concentration samples, take 400 µl of sample and mix with 100 µl of ice-cold PCA, place on ice for 5 min. Centrifuge at 13,000 x g for 2 min. Accurately transfer 380 µl of the supernatant to a fresh tube. Depending on the nature of the analyte, the samples in PCA may be frozen at -70°C for up to a month for storage at this stage.
2. Sample Neutralization:Add 20 µl of ice-cold Neutralization Solution (resuspend the fine precipitate) and mix to neutralize the sample and precipitate excess PCA. There may be some gas (CO2) evolution so vent the sample tube. Place on ice for 5 min. Spin briefly (~ 1 - 2 min). Samples are now deproteinized, neutralized, and PCA has been removed. The samples may now be used in a variety of assays directly.As you can see, the general ratio for 1 N PCA vs 6 N KOH is 5:1. But, I would recommend a pH check after neutralization to see if it is within the range of 6.5 to 8.0. If not, you might need to add more KOH.
Is the standard just ATP without any additives (like for example stabilizers)?
Yes, this is pure ATP w/o any additives.
I dont have liquid N2 or dry ice , if i want to store platelets to quick frozen for assay at later date and i have deep freezer in -70 C for this aim, is it possible ? If yes, how many days platelets can be store in this tempreture for later assay?When do i add lysing buffer to plateles, before freezing or afer defreezing when we wnat to assay?
I would not recommend storing the platelet lysate at -70°C since the ATP is very labile and would definitely need some liquid N2 or dry ice for storage. Therefore the best bet for this client is to freeze the platelets at -70°C and lyse them when he is ready to perform the assay.
I am looking for a method for analysing the ATP content in blood samples. Could this ATP colorimetric test kit be used for this purpose. If so, could you explain how the sample should be prepared and what volume I would require.
Yes, if you intend to use plasma or serum with this kit, it should definitely be compatible. In such a case, please deproteinize the sample as suggested in K808-200 kit protocol and then use the isolated fractions w/o any other pretreatment with these kits. You will have to do a pilot optimization assay in order to determine the best volume. If you plan on using whole blood, it might get slightly complicated. In such case we would need to make sure that you get rid of the red colour. Please use the Cat # 1997-25, 10 kD spin column for this purpose.
There are 2 ways you can efficiently lyse your cells for this assay kit. You can either homogenize the 1 x 10^6 cells with 100 ul of the assay buffer using 30-50 passes on ice, check for the homogenization and then deproteinate your samples with the K808-200 kit. Alternatively, you can just lyse your cells in the PCA using the protocol mentioned for the K808-200 kit, neutralize the lysate and use it in the wells. In this case you will have to use the assay buffer for diluting the samples in the wells.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kit's citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact yopur regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
How do I normalize my samples against protein concentration
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, I would strongly recommend you to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
Takasu, C. et al., (2017) Treatment with dimethyl fumarate ameliorates liver ischemia/reperfusion injury, World J Gastroenterol, Oct.2017, 23(25): 4508–4516.
Wu J et al., (2017) Redox imbalance and mitochondrial abnormalities in the diabetic lung, Redox Biol, 2017, 11:51-59
Johnstone M et al., (2017) Characterization of the Pro-Inflammatory Cytokine IL-1β on Butyrate Oxidation in Colorectal Cancer Cells, J Cellular Biochem, 2017, 118(6):1614-1621
Andres-Hernando, Ana et al. (2017) Protective role of fructokinase blockade in the pathogenesis of acute kidney injury in mice, Nat Commun. 2017 Feb 13;8:14181.
Rahn et al., Zebrafish lacking functional DNA polymerase gamma survive to juvenile stage, despite rapid and sustained mitochondrial DNA depletion, altered energetics and growth. Nucleic Acids Res., Oct 2015; 10.1093/nar/gkv1139.
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