製品の概要

  • 製品名
    Acetyl CoA Assay Kit
  • 検出方法
    Fluorescent
  • サンプルの種類
    Cell Lysate, Tissue Lysate
  • アッセイタイプ
    Quantitative
  • 検出感度
    > 0.01 nmol/well
  • 検出範囲
    0.01 nmol/well - 1 nmol/well
  • 全工程の試験時間
    0h 20m
  • 製品の概要

    Acetyl CoA Assay Kit ab87546 is a highly sensitive assay for quantifying Acetyl CoA level in  biological samples.


    In the Acetyl CoA assay protocol, free CoA is quenched then Acetyl CoA is converted to CoA. The CoA is reacted to form NADH which interacts with a probe to generate fluorescence (Ex=535/Em=587 nm).


    The assay can detect 10 to 1000 pmol of Acetyl CoA (with detection limit ~0.4 µM).


    Acetyl CoA assay protocol summary:
    - add samples and standards to wells
    - add CoA quencher to wells to remove background from free CoA and succ-CoA and incubate at room temp for 5 min
    - add quencher remover and incubate at room temp for 5 min
    - add reaction mix and incubate for 10 min at 37ºC
    - analyze with microplate reader

  • 特記事項

    This product was previously called PicoProbe Acetyl CoA Assay Kit.

    Acetyl CoA is a central molecule of metabolism. It carries acetate, used in the build-up and breakdown of larger molecules.

     

  • 試験プラットフォーム
    Microplate reader

製品の特性

画像

  • Induction of acetyl-CoA in mitochondria by palmitate. Mitochondria were isolated from cells after palmitate treatment (300 μM) at 1, 4 and 16 h, and then used in the concentration assay of acetyl-CoA. 

    The acetyl-CoA content was determined immediately after isolation of the mitochondria using ab87546. Briefly, acetyl-CoA standard curve was made in the range of 0–100 pM and the correlation coefficient was 0.990 or higher. Protein was removed in the sample using the perchloric acid protocol and the supernatant was neutralized with 3 M KHCO3. The CoASH Quencher and Quencher remover were added into the sample to correct the background generated by free CoASH and succ-CoA. The sample was then diluted with the reaction mix, and the fluorescence signal was measured at Ex/Em = 535/589 nm with Spectra max Gemini XPS (Molecular Devices, Sunnyvale, CA). The relative acetyl-CoA concentration was normalized with the mitochondrial protein.

  • Examples of standard curves generated following the ab87546 kit protocol.

プロトコール

参考文献

This product has been referenced in:
  • Jung TW  et al. ß-aminoisobutyric acid attenuates LPS-induced inflammation and insulin resistance in adipocytes through AMPK-mediated pathway. J Biomed Sci 25:27 (2018). Functional Studies . Read more (PubMed: 29592806) »
  • Ravera S  et al. Concentration-dependent metabolic effects of metformin in healthy and Fanconi anemia lymphoblast cells. J Cell Physiol 233:1736-1751 (2018). Read more (PubMed: 28681917) »
See all 16 Publications for this product

レビューと Q&A

1-10 of 19 Abreviews or Q&A

Answer

1.We recommend using ~2-5 X 10 ^ 6 cells for each manipulation.
2. Deproteinization is recommended for cells in culture. I have attached the protocol that the lab recommends for this step.

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Answer

In principle, eliminating the quenching and conversion step will allow you to measure CoA alone. However, the kit was not produced nor validated for this purpose, so we cannot guarantee that it will work, nor do we have a CoA standard that has been validated with this method to provide with the kit.



Alternatively, I would recommend one of our CoA detection kits listed below so you don't have to optimize and piece together the assay yourself.



Colorimetric or Fluorometric detection (detection range; 2.5 µM - 250 µM)

https://www.abcam.com/coenzyme-a-detection-kit-ab102504.html



Fluorometric detection (detection range: 40 nM - 10µM)

https://www.abcam.com/coenzyme-a-detection-kit-fluorometric-green-ab138889.html

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Answer



J’ai contacté le laboratoire concernant le kit ab87546 :

1.Nous recommandons d’utiliser ˜2-5 X 10^6 cellules pour chaque manip.

2. Il est recommandé de faire une déprotéinisation pour des cellules en culture. J’ai attaché le protocole que le laboratoire recommande pour cette étape.

3. Comme expliqué au téléphone, quand vous ajoutez le « CoA conversion » à vos échantillons, vous devez ajouter le mélange qui correspond à la gamme standard que vous utilisez. Par exemple, si vous pensez que vos échantillons sont faibles en acetyl CoA, utilisez la gamme et le mélange « CoA conversion » de 0-100 pmol.

4. Effectivement, il doit y avoir eu une inversion des tubes « Acetyl CoA Standard (1 µmol) (Lyophilised) » et « Acetyl CoA Enzyme Mix » et notre stock a maintenant été vérifié.

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Answer

1. Yes it is very crucial to do the sample prep exactly as recommended as Acetyl CoA is very sensitive to degradation and you risk the chance of inefficient results if you make changes to the protocol.
2. The deproteinization approach, though not as sensitive as the sample prep, would still benefit from using PCA rather than the 10 kDa spin filters. But you can use the spin filters if you do not have the option of using PCA.

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Answer

Thank you for contacting us.

I have had the opportunity to discuss the specificity of this kit with the lab as you have requested. I have found that in this particular assay the probe is specific to just the NADH.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you for your interest in our acetyl CoA assay kit, ab87546.

The assay measures relative amounts of acetyl CoA in samples indirectly, compared to a standard included with the kit, by converting the acetyl CoA to CoA, and then using the resulting CoA to reduce NAD to NADH, which in turn reacts with the probe to generate a fluorescent signal .

The conversion enzyme is what converts the acetyl CoA to CoA. The substrate mix is what the CoA acts upon, to generate the NADH. The components of the substrate mix and the identity of the conversion enzyme are considered proprietary information.

I hope this helps. Please let me now if you have any other questions.

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High background values in the fission yeast

Poor Average 3/5 (Ease of Use)
Abreviews
Protocol:
Two methods of sample preparation were tested.
1. Cell lysis by bead beating in acetyl-CoA assay buffer, deproteinization by 10 kDa spin column
2. Cell lysis by bead beating in TCA which also serves to denature proteins, neutralization by Na2CO3 to pH ~7
Extracts were prepared from fresh yeast biomass of S. pombe cells grown to exponential phase and processed as quickly as possible due to acetyl-CoA instability. Variable sample volumes in the assay - 5, 10 or 50 ul (samples diluted to 50 ul by assay buffer) were used. The standards and samples were measured in the 0-100 pmol range.

Figure legend:
A. Standard curve in the 0-100 pmol range. B. Fluorescence values of samples prepared from two yeast strains (A and B) using the 10kDa spin column. The acquired values of background samples (without conversion enzyme) are higher than those of samples. C. Fluorescence values of samples prepared from two yeast strains (A and B) using TCA precipitation. The acquired values of background samples is lower than those of samples only when 10 ul extract were used. Moreover, high volume (50 ul) of sample seems inhibitory for the assay.

Jarmila Tvarůžková

Verified customer

投稿 Feb 08 2018

Question
Answer

The sample can be stored at -80 degree centigrade once prepared. However, we recommend to use the sample immediately after preparation for maximum efficiency.

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Answer

I can confirm that kit PicoProbe Acetyl CoA Assay Kit (ab87546) can be used with frozen mouse tissues. Please see the protocol booklet for more information regarding sample preparation.

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Answer

1. Mitochonrial lysate can be assayed by ab87546.

2. For the amount sample to be added into each sample well, we recommended to perform a pilot experiment with a range of dilutions of the sample to determine the optimal concentration/dilution of sample which gives the best results in the linear range of the standard curve. Please note that the probe in this kit is a very sensitive probe (minimum detection level is 10 pmol of Acetyl CoA). You may start with 1 ul sample to 10ul sample per well.

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1-10 of 19 Abreviews or Q&A

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

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