製品の概要

  • 製品名
    NADP/NADPH Assay Kit (Fluorometric)
    NADP/NADPH キット 製品一覧
  • 検出方法
    Colorimetric/Fluorometric
  • サンプルの種類
    Cell culture extracts, Tissue Extracts
  • アッセイタイプ
    Quantitative
  • 全工程の試験時間
    1h 00m
  • 種交差性
    交差種: Mammals, Other species
  • 製品の概要

    NADP/NADPH Assay Kit (Fluorometric) ab176724 provides a convenient method for sensitive detection of NADP, NADPH and their ratio.


    The enzymes in the system specifically recognize NADP/NADPH in an enzyme recycling reaction that significantly increases detection sensitivity. In addition, this assay has very low background since it is run in the red visible range that considerably reduces the sample interference.


    The NADP/NADPH assay can be performed in a 96-well or 384-well microtiter-plate format. The signal can be read by either a fluorescence microplate reader at Ex/Em = 530-570/590-600 nm (maximum Ex/Em = 540/590 nm) or an absorbance microplate reader at ~576 nm.


    NADP/ NADPH assay protocol summary:
    - add samples and standards to wells
    - add extraction solutions and incubate for 15 min
    - add reaction mix and incubate for 30 min - 2 hr
    - analyze with microplate reader

  • 特記事項

    Nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) are two important cofactors found in cells. NADH is the reduced form of NAD+, and NAD+ is the oxidized form of NADH. NAD forms NADP with the addition of a phosphate group to the 2' position of the adenyl nucleotide through an ester linkage. NADP is used in anabolic biological reactions, such as fatty acid and nucleic acid synthesis, which require NADPH as a reducing agent. In chloroplasts, NADP is an oxidizing agent important in the preliminary reactions of photosynthesis. The NADPH produced by photosynthesis is used as reducing power for the biosynthetic reactions in the Calvin cycle of photosynthesis.

  • 試験プラットフォーム
    Microplate reader

製品の特性

  • 保存方法
    Store at -20°C. Please refer to protocols.
  • 内容 250 tests
    NADP Extraction Solution 1 x 10ml
    NADP/NADPH Control Solution 1 x 10ml
    NADP/NADPH Lysis Buffer 1 x 10ml
    NADP/NADPH Recycling Enzyme Mixture 2 vials
    NADPH Extraction Solution 1 x 10ml
    NADPH Sensor Buffer 1 x 20ml
    NADPH Standard 1 vial
  • 研究分野
  • 関連性
    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.

画像

  • Quantity of NADP, NADPH and total (NADP+NADPH) in U937 cells (duplicates; +/- SD). 0.5 x 107 cells were lysed in 1 mL of lysis buffer.

  • Quantity of NADP, NADPH and total (NADP+NADPH) in RAW 264.7 cells (duplicates; +/- SD). 0.5 x 107 cells were lysed in 1 mL lysis buffer.

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

プロトコール

参考文献

This product has been referenced in:
  • Layton CJ Diabetic levels of glucose increase cellular reducing equivalents but reduce survival in three models of 661W photoreceptor-like cell injury. BMC Ophthalmol 15:174 (2015). Read more (PubMed: 26653778) »
See 1 Publication for this product

レビューと Q&A

1-2 of 2 Abreviews or Q&A

NADPH measurement for mouse cell lines

Average Good 4/5 (Ease of Use)
Abreviews
The kit seems to work with mouse cell lines: min6b1 cells. But surprisingly Nadph quantity decreases with lower concentration of protein, and nadp is going in the opposite direction.
The protocole of the kit was not that clear ( it should be good to make an update of it) but the technical support is always ready to answer.

Abcam user community

Verified customer

投稿 Jan 17 2017

Answer

The lysis buffer is an addition for the kit and it is not essential that you use this lysis buffer to prepare your samples. Most of our customers who are using 96 well plate based on 25 uL/well find that 10 ml of lysis buffer is sufficient.

If you are using the tissues, you can dilute the samples with PBS for further assay. Do not use RIPA as a lysis buffer as this will interfere the assay. I would recommend you either use a traditional homogenization method, or use ab179835, Mammalian Cell Lysis Buffer 5X.

Please note that 250 assays is based on the assay formula of 25uL test samples + 25 μL of NADPH or NADP Extraction Solution + 25 μL of NADP or NADPH Extraction Solution + 75 uL NADP/NADPH reaction mixture.

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

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