Product Details
| abID | ab83359 |
|---|---|
| Cat # +Size | K355-100 |
| Size | 100 assays |
| Detection Method | Absorbance (570 nm) or Fluorescence (Ex/Em 535/587 nm) |
| Species Reactivity | Mammalian |
| Applications | In the assay, ADP is converted to ATP and pyruvate. The assay can detect as low as 1 µM ADP in biological samples. |
| Features & Benefits | • Simple procedure; takes less than 1 hour • Fast and convenient • The assay is sensitive, stable and high-throughput adaptable. |
| Kit Components | • ADP Assay Buffer • ADP Probe (lyophilized) • DMSO (anhydrous) • ADP Converter • ADP Developer Mix • ADP Standard (1 µmole lyophilized) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
ADP is a product of ATP dephosphorylation and it can be rephosphorylated to ATP. De-phosphorylation and rephosphorylation occur via various phosphatases, phosphorylases and kinases. ADP is stored in platelets and can be released to interact with a variety of purinergic receptors. ADP levels regulate several enzymes involved in intermediary metabolism. ADP conversion to ATP primarily occurs within the mitochondrion and chloroplast although several such processes occur in the cytoplasm. Conventionally, ADP levels are measured by luciferase/luciferin mediated assays after ADP is converted to ATP. However, the luciferase system is unstable and luminescence equipment is not generally available in most laboratories. BioVision’s newly designed ADP Assay Kit provides a convenient colorimetric and fluorometric means to measure ADP level. In the assay, ADP is converted to ATP and pyruvate. The generated pyruvate can be quantified by colorimetric (λmax = 570 nm) or fluorometric method (Ex/Em 535/587 nm). The assay is simple, sensitive, stable and high-throughput adaptable. The assay can detect as low as 1 µM ADP in biological samples.
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What are the differences between K355 and K356?
There are 3 critical differences between these 2.Sample type: K355 : For samples having strong absorbance at 450 nm, having NADH recycling system or high Gluose-6-Phosphate content. K356: For samples having high levels of pyruvate or reducing substances, which may interfere with oxidase-based assay.Detection Method: K355: Colorimetric (?max = 570 nm) or fluorometric (Ex/Em = 535/587 nm). K356: Colorimetric (?max = 450 nm).Detection limit: As low as 1 µM ADP in biological samples. K356: Less than 20 µM ADP in biological samples.
Does 2.5% perchloric acid interfere with the ADP assay kit? I would like to pre-treat my samples with 2.5% perchloric acid to remove protein prior to testing for ADP.
I would recommend the spin filter over the PCA treatment. Theoretically the PCA treatment should be fine, but since we have not tested the outcome of this assay with such treated samples, I would go with the spin filter to be sure of no adverse effects.
The customer needs to freeze the samples before using the ADP and ATP kits K354-100 and K355-100. They do however not have the columns to deproteinate them beforehands and are not able to order them in time. The question now is what alternative methods the customer could use. Can the customer prepare the samples in perchloric acid or TCA for use in both kits? Would the preparation in perchloric acid or TCA still allow compatiblity with the ADP kit?
Both the assay buffers are different. So the samples have to be made individually for both assays. For the K354-100 kit, samples lysed in the ATP assay buffer should be quick frozen using liquid N2 or dry ice if it is to be assayed at a later date. Samples can either be deproteinized or spin filtered. You client does not have the filter. So it has to be deproteination. Follow the protocol of K808-200 for this. The neutralization is with 6 N KOH.For K355-100 samples lysed in the ADP assay buffer can be aliquoted and frozen at -80°C before use. Do not filter or deproteinate these samples.
Our customer would like to know about K355-100:She has sample lysates prepared in Tris, EDTA.1) Are these compatible with the kit?2) Can those samples be diluted in assay buffer or does she need to prepare the samples again just in assay buffer?
For most efficient results, I always recommend that the samples be made in the assay buffer provided with the kit. In case of that not being possible due to sample limitations, you can dilute it in the assay buffer as a last resort. However, this might compromise the results to a certain extent.
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 your 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.
We bought both K354 and K355. Can we use the same assay buffer to prepare samples for both kit?
Unfortunately, the composition of the assay buffers is different in K354 and K355. Therefore, you cannot use the same buffer to prepare samples for the 2 kits. We recommend using the assay buffer that is provided with each kit to prepare the samples for the respective assays.
Zhang et al., Reduced autophagy in livers of fasted, fat-depleted, ghrelin-deficient mice: Reversal by growth hormone. PNAS, Jan 2015; 112: 1226 - 1231.
Ying Liu et al., Adiponectin Corrects High-Fat Diet–Induced Disturbances in Muscle Metabolomic Profile and Whole-Body Glucose Homeostasis. Diabetes, Mar 2013; 62: 743 - 752.
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