Overview

  • Product name

    NADP/NADPH Assay Kit
    See all NADP/NADPH kits
  • Detection method

    Colorimetric
  • Sample type

    Tissue Extracts, Cell Lysate
  • Assay type

    Quantitative
  • Assay time

    2h 00m
  • Product overview

    NADP/NADPH Assay Kit (ab65349) provides a convenient tool for sensitive detection of the intracellular nucleotides: NADP, NADPH and their ratio. Assays of nicotinamide nucleotides are of continual interest in the studies of energy transforming and redox state of cells or tissue.


    The enzymes in the system specifically recognize NADP/NADPH in an enzyme cycling reaction. The assay does not recognize NAD+/NADH. There is no need to purify NADP/NADPH from the sample mix. The enzyme cycling reaction significantly increases detection sensitivity. Results can be quantified using a plate reader at OD450nm.


    NADP / NADPH assay protocol summary:
    - extract samples from cells /  tissues with extraction buffer and deproteinize with spin column
    - for NADPH measurement, heat samples to 60ºC for 30 min to decompose NAD+, cool on ice (this step not necessary for measurement of total NADP+/NADPH)
    - add samples and standards to wells
    - add reaction mix and incubate for 5 min at room temp to convert NADP to NADPH
    - add NADPH developer and incubate for 1-4 hrs while reaction cycles
    - analyze with microplate reader multiple times during the 1-4 hr incubation
    - reaction can be stopped with stop solution
     

  • Notes

    If you would like to use a fluorometric reading, please refer to NADP/NADPH Assay Kit (Fluorometric) (ab176724).

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components Identifier 100 tests
    NADP Cycling Buffer WM 1 x 15ml
    NADP Cycling Enzyme Mix Green 1 x 0.2ml
    NADP/NADPH Extraction Buffer NM 1 x 50ml
    NADPH Developer Purple 1 vial
    NADPH Standard (MW:833.36) Yellow 1 x 166.7µg
    Stop Solution Red 1 x 1.2ml
  • Research areas

  • Relevance

    NADP (Nicotinamide adenine dinucleotide phosphate) is a coenzyme composed of ribosylnicotinamide 5-phosphate (NMN) coupled by pyrophosphate linkage to the 5-phosphate adenosine 2,5-biphosphate. It serves as an electron carrier in a number of reactions, being alternately oxidised (NADP+) and reduced (NADPH). The oxidative phase of the pentose phosphate pathway is the major source of NADPH in cells, producing approxiamtely 60% of the NADPH required. NADPH provides the reducing equivalents for biosynthetic reactions and the oxidation-reduction involved in protecting against the toxicity of ROS, allowing the regeneration of GSH. NADPH is also used for anabolic pathways, such as lipid synthesis, cholesterol synthesis and fatty acid chain elongation.

Images

  • NAPDH/NADP+ ratio was determined using ab65349 in stable HSPB1- knockdown U87 cells. 

  • Standard curve with background signal subtracted (duplicates; +/- SD).

  • Total NADP and NADPH (tNADP) or NADPH alone measured in RAW cell lysates (duplicates +/- SD).

  • Measurement of NADPt and NADPH in rat liver lysate (20 μg). Assays were performed using the kit protocol.

  • Measurement of NADPt and NADPH in HeLa cell lysate (80 μg). Assays were performed using the kit protocol.

  • Typical standard curve for ab65349.

Protocols

References

This product has been referenced in:

  • Pan X  et al. H2Se Induces Reductive Stress in HepG2 Cells and Activates Cell Autophagy by Regulating the Redox of HMGB1 Protein under Hypoxia. Theranostics 9:1794-1808 (2019). Read more (PubMed: 31037139) »
  • Chen L  et al. LncRNA GAS5 regulates redox balance and dysregulates the cell cycle and apoptosis in malignant melanoma cells. J Cancer Res Clin Oncol 145:637-652 (2019). Read more (PubMed: 30569211) »
See all 41 Publications for this product

Customer reviews and Q&As

1-10 of 62 Abreviews or Q&A

Abreviews
We used the kit to quantify the levels of NADPH and NADP in BV2 microglia cells. Cells in the first experiment were treated with LPA (100ng/ml). The kit worked fine and we will proceed with the experiments studying the impact of our substance of interest on NADPH/NAPH levels.
Be aware that the columns that are needed during the procedure are not provided with the kit. They need to be ordered separately.

Dr. Lisha Joshi

Verified customer

Submitted Jun 13 2019

Answer

We highly recommend to follow storage instructions on the individual datasheets.  For ab65349 kit it should be stored at -80oC when it is delivered and for short term storage.

It is also worth noting that the components of some kits, once reconstituted, should be stored at different storage conditions.  For this kit, some of the reconstituted components need to be stored at -80oC and are stable for 2 months only.  Please see the protocol booklet for details, page 8 section 9:

NADPH Standard:

Reconstitute NADPH Standard with 200 μL of pure DMSO to generate a 1 nmol/μL (1mM) NADPH Standard solution. Aliquot standard so that you have enough volume to perform the desired number of assays. Store aliquots at -80°C. Use within two months.

NADPH Developer:

Reconstitute NADPH Developer in 1.2ml of ddH2O. Pipette up and down several times to completely dissolve the pellet in solution. Do not vortex. Aliquot so that you have enough volume to perform the desired number of experiments. Store at -80°C. Use within two months.

NADP Cycling Enzyme Mix:

Ready to use as supplied. Keep on ice protected from light during the assay. Aliquot so that you have enough volume to perform the desired number of assays. Store aliquots at -80°C.

NADPH Cycling Buffer:

Ready to use as supplier. Equilibrate to room temperature before use. Store at 4°C.

NADP/NADPH Extraction Buffer:

Ready to use as supplier Equilibrate to room temperature before use. Store at 4°C.


Stop Solution:

Ready to use as supplier Equilibrate to room temperature before use. Store at 4°C.

Link to the protocol booklet:

https://www.abcam.com/ps/products/65/ab65349/documents/ab65349%20NADP%20NADPH%20Assay%20kit%20protocol_v18%20(website).pdf


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Question
Answer


I am sorry we have not yet tested ab65349 NADP/NADPH Assay Kit in serum samples so we do not have laboratory information to provide to help answer your question. All tested sample types covered by the Abpromise guarantee are listed on the individual kit datasheets.


As far as I am aware, no special pretreatment would be required, but you may want to check with a literature search what level of NADP/NADPH you would expect to find in serum and if this falls within range of the assay.  If you wanted to try testing serum I would suggest to test directly by adding sample to the measuring wells. In order to find the optimal sample dilution, we recommend running a small pilot experiment with a few representative samples at different dilutions.


I appreciate that using a kit that is not tested for serum samples may pose some risk, so if the reading are expected to be in range of the assay I would like to invite you to our AbTrial program. By participating in our AbTrial program you can use this product without financial risk by simply sharing your results for ab65349NADP/NADPH Assay Kit in an Abreview.


How to take part in our AbTrial program:


1. Purchase the product at regular price and reply to this email with your PO or order number.


2. I will generate a personal discount code equal to the value of the product.


3. Submit your results (including an image) at www.abcam.com/abreview. Enter your discount code in the additional notes section of the AbReview.


4. Once the AbReview is submitted, the discount code will become active.


5. Use your discount code to receive that value off your next purchase.


The Terms and Conditions of this offer can be found at www.abcam.com/abtrial.


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Answer

Thank you for your enquiry.
I have now heard back from the laboratories. They have confirmed that they have not tested this kit with samples stored in RNAlater but you can remove the RNA later solution and use the extraction buffer to prepare the samples.

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Question
Answer


1. Some liquid loss during heating (can happen even in tubes if the liquid is not spun down). Be sure to cool and spin down the tubes following decomposition.

2. Endogenous enzymes converting to NADPH if the samples are not deproteinized.
If you are not already deproteinizing your samples the endogenous enzymes may be converting to NADPH as the lab suggested below, in which case we recommend our 10 kD spin columns ab93369 (link below)

https://www.abcam.com/10kd-spin-column-ab93349.html

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Question
Answer


NADPH oxidase is typically a complex made up of a cluster of proteins that donate an electron from NADPH to molecular oxygen (O2) to produce superoxide (O2-). This enzyme activity is not measured by this kit directly.


Due to NADPH oxidation, NADP will be generated and the concomitant decrease in NADPH accompanied by a proportional increase in NADP can be measured by this kit.



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Answer

Below is our recommended procedure. The key is to keep your samples on ice during homogenization to avoid heating and degradation of the analyte and to ensure lysis under the microsope before proceding with the assay.


General Sample preparation guidelines

For cells and tissues:

- Start with 1-2X106 cells / 10-100mg tissue per assay

- Add ˜4 volumes of the assay buffer/cell lysis buffer on ice

- Homogenize using a Dounce homogenizer (10-50 passes) on ice

- Confirm lysis by viewing an aliquot under the microscope

- Spin down, collect supernatant.

- Load the supernatant unto a 10kda spin column for deproteinzation (if indicated on the data sheet; not for enzyme assays)

- Use the eluate for subsequent assays.

Notes:

- Extraction buffers: incubate cells with buffer on ice

- Appropriate dilutions of the sample must be tested in order ensure the readings will fall within the linear range of the standard curve.

- Some data sheets specifically mention to prepare cell lysates by freeze/ thaw cycles. Please follow the instruction on the data sheet for sample preparation if it is mentioned.

- Micro-sonicator can be used to enable better cell lysis

- Samples on ice in order to avoid sample heating

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Answer

If you suspect insufficient lysis, I recommend taking a small volume and visualizing under a microscope to ensure that the cell membrane has been lysed. If not, you can try an additional freeze-thaw cycle, or try homogenizing with a dounce homogenizer.

We recommend a 10 kDa spin column but do not require it. If you are strict about performing your assay quickly after sample preparation and keeping your sample on ice as much as possible you may be able to avoid degradation.

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Answer

One modification to the protocol that may help is the following:
Seal the plate and then heat at 60C for 30 mins. During this time evaporation reduces the volume and hence makes the solution more concentrated which is why often the NADPH OD is greater than NADP total OD.
Once the heating is done put plate on ice for a few minutes and then centrifuge carefully. These steps should help with this issue.

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Answer



I double checked and unfortunately, have confirm that we do not have a kit for measuring NADPH oxidase in our catalog at the moment. We do offer ab65349 which is a NADP/NADPH Assay Kit:

https://www.abcam.com/index.html?datasheet=65349 (or use the following: https://www.abcam.com/index.html?datasheet=65349).

I can recommend the following products

ab118970 (Lipid Peroxidation (MDA) Assay Kit)

https://www.abcam.com/index.html?datasheet=118970 (or use the following: https://www.abcam.com/index.html?datasheet=118970).

ab139473 (Cellular ROS/RNS Detection Assay Kit)

https://www.abcam.com/index.html?datasheet=139473 (or use the following: https://www.abcam.com/index.html?datasheet=139473).

ab139476 (Cellular ROS/Superoxide Detection Assay Kit)

https://www.abcam.com/index.html?datasheet=139476 (or use the following: https://www.abcam.com/index.html?datasheet=139476).

ab139473, Cellular ROS/RNS Detection Assay Kit for microscopy is used for monitoring reactive oxygen and/or nitrogen species (ROS/RNS) in live cells.

This kit can be used for discriminates among superoxide, nitric oxide and peroxynitrite by fluorescence microscopy. The kit includes three fluorescent dye reagents.

ab139476, Cellular ROS/Superoxide Detection Assay Kit: Total ROS/Superoxide detection kit is designed to directly monitor real time reactive oxygen species (ROS) production in live cells using fluorescence microscopy or flow cytometry. The kit includes two fluorescent dyes as major components: Oxidative Stress Detection Reagent (Green) for total ROS detection reagent and Superoxide Detection Reagent (Orange).

This kit distinguishes between different reactive species, such as hydrogen peroxide, peroxynitrite and hydroxyl radicals.

I can confirm that all our kits are covered by our Abpromise guarantee for m12 month if they are unopened and stored as instructed in the protocol booklet. Please note that some reagents in the kits can only be stored short term if they are opened or reconstituted.

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1-10 of 62 Abreviews or Q&A

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