概述

  • 产品名称NADP/NADPH Assay试剂盒
    参阅全部 NADP/NADPH 试剂盒
  • 检测方法Colorimetric
  • 测试
    100 x 1 assay
  • 样品类型
    Tissue Extracts, Cell Lysate
  • 检测类型Quantitative
  • 检测时间
    2h 00m
  • 产品概述

    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 (It does not recognize NAD+/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 OD450nm.
    Visit our FAQs page for tips and troubleshooting.

     

  • 说明

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

  • 经测试应用适用于: Functional Studiesmore details
  • 平台Microplate

性能

  • 相关性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.

应用

Our Abpromise guarantee covers the use of ab65349 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

应用 Ab评论 说明
Functional Studies Use at an assay dependent dilution.

NADP/NADPH Assay Kit 图像

  • 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.

实验方案

NADP/NADPH Assay Kit (ab65349)参考文献

This product has been referenced in:
  • Ogrunc M  et al. Oncogene-induced reactive oxygen species fuel hyperproliferation and DNA damage response activation. Cell Death Differ 21:998-1012 (2014). Functional Studies . Read more (PubMed: 24583638) »
  • Sun Y  et al. Estradiol promotes pentose phosphate pathway addiction and cell survival via reactivation of Akt in mTORC1 hyperactive cells. Cell Death Dis 5:e1231 (2014). Read more (PubMed: 24832603) »

See all 16 Publications for this product

Product Wall

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 s...

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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 deproteinize...

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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 ox...

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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 prepa...

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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....

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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 gre...

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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:

http://www.abcam.com/index.html?datasheet=6534...

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We recommend using the buffers provided with the specific kits for our assays. So, unfortunately we don’t suggest using the same buffer for both assays (ab65348 and ab65349).

It is very likely that cell or tissue lysates contain enzymes that consume NADPH rapidly. If the cells are already lysed in RIPA, I am assuming that they have not been deproteinized. Therefore I would not recommend using these lysates. Please make fres...

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Yes, the extraction buffers from ab65348 and ab65349 are the same.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"