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Product Details
| Cat # +Size | K337-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Absorbance (450 nm) |
| Species Reactivity | Mammalian |
| Applications | BioVision’s NADH/NAD Quantification Kit provides a convenient tool for sensitive detection of the intracellular nucleotides: NADH, NAD and their ratio. |
| Features & Benefits | • Simple procedure; takes ~2 hours • Fast and convenient • Kit contains the necessary reagents for accurate measurement of NAD and NADH and their ratio. |
| Kit Components | • NADH/NAD Extraction Buffer • NAD Cycling Buffer • NAD Cycling Enzyme Mix • NADH Developer • Stop Solution • NADH Standard (MW:763) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Assay of nicotinamide nucleotides is of continual interest in the studies of energy transforming and redox state of cells or tissues. BioVision’s NADH/NAD Quantification Kit provides a convenient tool for sensitive detection of the intracellular nucleotides: NADH, NAD and their ratio. The NAD Cycling Enzyme Mix in the kit specifically recognizes NADH/NAD in an enzyme cycling reaction. There is no requirement to purify NADH/NAD from samples. The reaction specifically detects NADH and NAD, but not NADP nor NADPH. The enzyme cycling reaction significantly increases the detection sensitivity and specificity. NADt (NAD and NADH) or NADH can be easily quantified by comparing with standard NADH.
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We want to do tissue deproteinizing with K808-200 first for tissue samples and then use K337-100. Is it ok?
Since there is concern about compromised NADH quantitation after exposure to very acidic pH from kit K808-100, we strongly recommend using the 10 kD spin filters (Cat # 1997-25) for sample deproteinization.
We would like to use yeast cells with this kit. Do you have the protocol?
We have optimized this assay for mammalian cells. It has not been tested with yeast cells. But the extraction buffer cab be modified slightly to work efficiently with yeast cells. Since we have not used yeast cells, we cannot comment on the exact modifications , but here is a nice comprehensive abstract we found which could be helpful in determining those:
Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis.
Harder A, Wildgruber R, Nawrocki A, Fey SJ, Larsen PM, Gorg A.
Source
Technical University of Munich, Department of Food Technology, Freising-Weihenstephan, Germany.
Abstract
Three different procedures for the solubilization of yeast (S. cerevisiae) cell proteins were compared on the basis of the obtained two-dimensional (2-D) polypeptide patterns. Major emphasis was laid on minimizing handling steps, protein modification or degradation, and quantitative loss of high molecular mass proteins. The procedures employed were sonication, followed by (i) protein solubilization with "standard" lysis buffer (9 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), 2% v/v carrier ampholytes, (ii) presolubilization of proteins with sodium dodecyl sulfate (SDS) buffer, consisting of 1% SDS and 100 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.0, followed by dilution with "standard" lysis buffer, and (iii) boiling the sample with SDS during cell lysis, followed by dilution with thiourea/urea lysis buffer (2 M thiourea/ 7 M urea, 4% w/v CHAPS, 1% w/v DTT, 2% v/v carrier ampholytes). All procedures tested were rapid and simple. However, with the first procedure (i), considerable degradation of high Mr proteins occurred. In contrast, protein degradation was minimized by boiling the sample in SDS buffer immediately after sonication (method ii). Protein disaggregation and solubilization of high Mr proteins were further improved by pre-boiling with SDS and using thiourea/urea lysis buffer instead of "standard" lysis buffer (procedure iii).
Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis.
Harder A, Wildgruber R, Nawrocki A, Fey SJ, Larsen PM, Gorg A.
Source
Technical University of Munich, Department of Food Technology, Freising-Weihenstephan, Germany.
Abstract
Three different procedures for the solubilization of yeast (S. cerevisiae) cell proteins were compared on the basis of the obtained two-dimensional (2-D) polypeptide patterns. Major emphasis was laid on minimizing handling steps, protein modification or degradation, and quantitative loss of high molecular mass proteins. The procedures employed were sonication, followed by (i) protein solubilization with "standard" lysis buffer (9 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), 2% v/v carrier ampholytes, (ii) presolubilization of proteins with sodium dodecyl sulfate (SDS) buffer, consisting of 1% SDS and 100 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.0, followed by dilution with "standard" lysis buffer, and (iii) boiling the sample with SDS during cell lysis, followed by dilution with thiourea/urea lysis buffer (2 M thiourea/ 7 M urea, 4% w/v CHAPS, 1% w/v DTT, 2% v/v carrier ampholytes). All procedures tested were rapid and simple. However, with the first procedure (i), considerable degradation of high Mr proteins occurred. In contrast, protein degradation was minimized by boiling the sample in SDS buffer immediately after sonication (method ii). Protein disaggregation and solubilization of high Mr proteins were further improved by pre-boiling with SDS and using thiourea/urea lysis buffer instead of "standard" lysis buffer (procedure iii).
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.
In the protocol, it is mentionned that to detect NADH, the NAD needs to be decomposed before the reaction. Does it mean that 1) there is no NAD present in the well, and 2) if the NAD cycling mix is added at this step, no reaction should be observed? Could you also summarize the way the kit works? How and when is the NAD measured? How and when is the NADt quantified?
This kit runs on a simple principle. You can either measure the total NAD+NADH or just the NADH. To measure the NAD, you need to subtract NADH levels from the NADt levels. This kit will only measure NADH. So if NADt has to measure, all the existing NAD has to be converted to NADH prior to detection. This is what the cycling enzyme does. If the levels of NADH only are to be measured, the NAD needs to be decomposed, which is done at an elevated temp.
I bought the kit and have another question concerning the protocol. It says, for the detection of NADH I need to do the decomposing step on 60 C first. The next step would be the NAD Cycling, where NAD is transformed into NADH. The protocol does not mention anything about leaving this step out if I only want to detect NADH. I guess it is useless if I decomposed NAD in the step before? So after decomposing NAD I'd directly jump to adding the developer, is that correct? Another question, the protocol says incubation for 1 to 4 hours: will reaction results after 1 hour differ much from results after 4 hours? I don't understand how long I have to incubate the samples with the developer to have my end-results.
It seems logical to ignore the step of NAD conversion to NADH if you have already decomposed the NAD, but I would still recommend you to follow the protocol exactly without deleting any steps. The NAD conversion to NADH adds some volume to each of the samples, as well as the standards. You definitely want this volume to be consistent between the standards and samples for comparing between them. Therefore, please do all the steps even if you want to assay just for NADH.
Your signals from 1 to 4 hrs of final incubation will ideally increase. Within that time range, whenever you are comfortable and satisfied with the signals, you can add the stop solution to terminate any more colour development.
Your signals from 1 to 4 hrs of final incubation will ideally increase. Within that time range, whenever you are comfortable and satisfied with the signals, you can add the stop solution to terminate any more colour development.
I would like to know something more specific about the reactions, namely what exactly is happening in the cycling reaction. From what I understand, at that time we will have two different tubes for the same sample, one for NADt and one for NADH. What is the function of the NAD cycling mix?
In the cycling reaction, all the NAD is getting converted to NADH. The NAD cycling mix will help in the conversion of NAD to NADH. Note that you can detect only NADH in this assay, since there is no NAD developer included. Therefore to measure total NADH, you will need to convert the existing NAD to NADH, and then when you develop the reaction, you will get the total NADH. To measure only NAD, you will decompose the NAD, measure for NADH and then subtract that from the total NADH reading.
Stop solution, should it be added before or after the final measurement? Usually the stop solution is added before, so all the reactions stop at the same, and the measurements are not bias. However, the way it is written in the protocol, it seems the readings are done before the stop solution is added.
Yes, the stop solution is added before the final measurement. You just keep developing the color until it falls within the linear range of the standard curve (which you can do only when you measure the absorbance). Once this colour is reached, you add the stop solution to all wells and then take the final measurement. Thus there is no measurement bias introduced.If we choose to lyse cells by homogenizing instead of freeze-thaw, what kind of device or method is the most suitable?
Please use a Dounce homogenizer. About 30-50 passages should be good for the homogenization. You can perform a microscopic examination to ascertain the homogenization. If required, please do 10-20 more passages.
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.
How can we use serum for this assay? Can you please share the protocol for measuring both NADt and NADH?
Serum samples need not be processed but can be used directly for the assay. This means you can collect serum and use it directly to the wells of a 96-well plate for the assay.
For serum samples, although NADH consuming enzymes like LDH are present only in minute quantities, we suggest removing any enzyme that may consume NADH by filtering the serum through a 10 kDa spin filter (BV Cat# 1997) and using the flow through for the assay. Please do a pilot study to assess how much serum you need for the assay. You can try different sample volumes to ensure the readings are within the linear range of the standard curve.
You can measure two things with this assay, NADt, which is NAD+NADH and for this, you can directly use the filtered serum.
For measuring NADH, you need to decompose the existing NAD. Please place the serum in a tube and heat it at 60°C for 25-30 min. If serum proteins precipitate, centrifuge and take the supernatant for the assay. Heating will decompose all NAD and the serum will now have only NADH. Serum is now ready to be used for measuring NADH.
For serum samples, although NADH consuming enzymes like LDH are present only in minute quantities, we suggest removing any enzyme that may consume NADH by filtering the serum through a 10 kDa spin filter (BV Cat# 1997) and using the flow through for the assay. Please do a pilot study to assess how much serum you need for the assay. You can try different sample volumes to ensure the readings are within the linear range of the standard curve.
You can measure two things with this assay, NADt, which is NAD+NADH and for this, you can directly use the filtered serum.
For measuring NADH, you need to decompose the existing NAD. Please place the serum in a tube and heat it at 60°C for 25-30 min. If serum proteins precipitate, centrifuge and take the supernatant for the assay. Heating will decompose all NAD and the serum will now have only NADH. Serum is now ready to be used for measuring NADH.
Adam L. Orr, Neuronal Apolipoprotein E4 Expression Results in Proteome-Wide Alterations and Compromises Bioenergetic Capacity by Disrupting Mitochondrial Function. J Alzheimers Dis, April 2019; 30883359.
Chloé Najac, In vivo investigation of hyperpolarized [1,3-13C2]acetoacetate as a metabolic probe in normal brain and in glioma. Sci Rep., March 2019; 30833594.
Grigorij Schleifer, Impaired hypoxic pulmonary vasoconstriction in a mouse model of Leigh syndrome. Am J Physiol Lung Cell Mol Physiol., Feb 2019; 30520688.
Don Benjamin, Dual Inhibition of the Lactate Transporters MCT1 and MCT4 Is Synthetic Lethal with Metformin due to NAD+ Depletion in Cancer Cells. Cell Rep, Dec 2018; 30540938.
Le Li, TAp73-induced phosphofructokinase-1 transcription promotes the Warburg effect and enhances cell proliferation. Nat Commun, Nov 2018; 30409970.
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