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.

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参考文献

This product has been referenced in:

See all 24 Publications for this product

レビューと Q&A

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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

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