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Product Details
| Cat # +Size | K347-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Absorbance (450 nm) |
| Species Reactivity | Mammalian |
| Applications | Convenient tool for sensitive detection of the intracellular nucleotides: NADP, NADPH and their ratio |
| Features & Benefits | • Simple procedure; takes ~2 hours • Fast and convenient • Kit contains the necessary reagents for accurate measurement of NADP and NADPH and their ratio • Enzymes in the system specifically recognize NADP/NADPH in an enzyme cycling reaction (It does not recognize NADᶧ/NADH) |
| Kit Components | • NADP/NADPH Extraction Buffer • NADP Cycling Buffer • NADP Cycling Enzyme Mix • NADPH Developer • Stop Solution • NADPH Standard (MW:833.36) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Assays of nicotinamide nucleotides are of continual interest in the studies of energy transforming and redox state of cells or tissue. The NADP/NADPH Quantification Kit provides a convenient tool for sensitive detection of the intracellular nucleotides: NADP, NADPH and their ratio. The enzymes in the system specifically recognize NADP/NADPH in an enzyme cycling reaction (It does not recognize NADᶧ/NADH). There is no need to purify NADP/NADPH from sample mix. The enzyme cycling reaction significantly increases detection sensitivity. Results can be quantified using plate reader at OD450 nm.
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Can we use this protocol with bacterial cells?
We have optimized the protocol for mammalian cells. Therefore, although I do not have data from bacterial samples for this, I would suggest the use of more number of starting cells, since bacterial cells are smaller. Additionally our extraction buffer may be fine with gram negative bacteria, but positive bacteria might need some manipulations. The protocol might have to be optimized for fitting bacterial samples
Are the extraction buffers in k337 and k347 the same?
Yes, these buffers are the same.
Our NEAT samples are showing the lowest concentration of NADP and the concentration increased with each dilution. The 1:100 dilution showed a higher reading than the sample diluted 1:10 in reaction buffer. Why?
In this case it is possible that the sample has an inhibitory substance. Therefore, increasing the sample concentration is correspondingly resulting in lower readings.
When I have done NADPH and NADH assay, I used extraction buffer for NADH kit as you advised me below. By the way, the result showed NADPH only value is higher than total NADP (NADP+ NADPH). NADH looked fine. I made NADPH by heating up at 60C for 30 min as described at protocol. Based on my knowledge total NADPH should be higher than NADPH only, right? Whats wrong with my result?
I think that in the heating process to decompose NADP there must have been some evaporation that made the resulting solution more concentrated, which might lead to this problem. Do the heating in a sealed container.
How do we normalize the number of cells? Do we first estimate the protein concentration and then do the assay or first do assay then do normalisation? kindly tell me how to normalize the readings.
For the NAD/NADH (k337) and the NADP/NADPH quantification kit (K347), you are taking a specific number of cells for the assay and doing the protein estimation and the assay in parallel or independant of each other. Use the same number of cells as used for the assay in order to do the protein analysis. For the assays use freeze/thaw two cycles (20 min on dry-ice, then 10 min at room temperature), or Dounce homogenization.
We are planning on grounding the sample at -80C to powder. I wonders if I still need to homogenise the sample after adding NADP/NADPH Extraction Buffer. Is BCA suitable for protein quantitation?
In such case, please resuspend the powder in the assay buffer, extract out the NADH/NADPH and quickly deproteinate the samples. Then continue with the recommended protocol. Yes, BCA estimation can be used.
Product protocol step No. C.3. (Incubate the plate at room temperature for 5 min to convert NADP to NADPH). In this step, all NADP is converted to NADPH?
Yes, if there is any NADP in the samples, it will get converted to NADPH in the step C3 of this protocol.
Product protocol step No. C.4. (Add 10 ml NADPH developer into each well. Let the reaction develop for 1 to 4 hours.). I measured the samples absorbance after 24 hours. The samples absorbance is higher than 24 hours before. Is this normal? In this step, what kind of reaction has occurred?
In step C4 the NADPH reacts with the probe in the developer to produce a colour. I am not sure what you mean by abs after 24 hrs. Did you perform the incubation for 24 hrs before you added the stop solution? If you incubated for that long, then yes, there will be color development. But if you added the stop solution and 24 hrs after that also you saw increased colour then it is unusual. The bottom line is that you add the stop solution once the absorbance falls within the linear range of the standard curve. You can then take a reading within 48 hrs of adding the stop solution, since the colour is stable for that long.
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 -20C. 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 have used this kit with 3D culture in Matrigel, but it does not seem to work. Do you have any recommendations?
Unfortunately, we have not tested this kit on matrigel 3D culture. This kit is meant to be used for regular cells and tissues. That being said, we assume that you would need to release the cells from the matrigel through a digestion step before using it with this kit.
Hu J et al., (2017) Tumor grade related expression of neuroglobin is negatively regulated by PPARγ and confers antioxidant activity in glioma progression, Redox Biol, 2017, 12:682-689
Wu J et al., (2017) Redox imbalance and mitochondrial abnormalities in the diabetic lung, Redox Biol, 2017, 11:51-59
Chaman et al., ERK2-Pyruvate Kinase Axis Permits Phorbol 12-Myristate 13-Acetate-induced Megakaryocyte Differentiation in K562 Cells. J. Biol. Chem., Sep 2015; 290: 23803 - 23815.
Zhao et al., Regenerative Neurogenesis After Ischemic Stroke Promoted by Nicotinamide Phosphoribosyltransferase–Nicotinamide Adenine Dinucleotide Cascade. Stroke, Jul 2015; 46: 1966 - 1974.
Li et al., Mitofusin-2-mediated tethering of mitochondria and endoplasmic reticulum promotes cell cycle arrest of vascular smooth muscle cells in G0/G1 phase. Acta Biochim Biophys Sin, Jun 2015; 47: 441 - 450.
For more citations of this product click here
