PicoProbe Acetyl CoA Assay试剂盒(ab87546)

概述

  • 产品名称
    PicoProbe Acetyl CoA Assay试剂盒
  • 样品类型
    Cell Lysate, Tissue Lysate
  • 检测类型
    Quantitative
  • 灵敏度
    > 0.01 nmol/well
  • 范围
    0.01 nmol/well - 1 nmol/well
  • 产品概述

    Abcam's PicoProbe Acetyl CoA Assay Kit is a highly sensitive assay for determining Acetyl CoA level in a variety of biological samples. In the assay, free CoA is quenched then Acetyl CoA is converted to CoA. The CoA is reacted to form NADH which interacts with PicoProbe 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) in a variety of samples.
    Visit our FAQs page for tips and troubleshooting.

  • 说明

    Acetyl CoA is a central molecule of metabolism. It carries acetate, used in the build-up and breakdown of larger molecules. Acetyl CoA is key in synthetic pathways leading to sesquiterpenes, precursors to cholesterol and other sterols, flavenoids and other polyketides, polyenes and long-chain fatty acids. It is the source of the acetyl group used in histone acetylation. The acetyl group is also incorporated into a variety of other molecules such as acetylcholine, melatonin, heme and TCA cycle intermediates.

  • 经测试应用
    适用于: Functional Studiesmore details

性能

应用

Our Abpromise guarantee covers the use of ab87546 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.

图片

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

实验方案

文献

This product has been referenced in:
  • Dai X  et al. A novel small-molecule arylsulfonamide causes energetic stress and suppresses breast and lung tumor growth and metastasis. Oncotarget 8:99245-99260 (2017). Read more (PubMed: 29245898) »
  • Gopal U & Pizzo SV Cell surface GRP78 promotes tumor cell histone acetylation through metabolic reprogramming: a mechanism which modulates the Warburg effect. Oncotarget 8:107947-107963 (2017). Read more (PubMed: 29296215) »

See all 11 Publications for this product

客户评价及客户问答

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.

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 val...

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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 ...

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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 a...

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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 ...

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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 ...

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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.
Username

Jarmila Tvarůžková

Verified customer

提交于 Feb 08 2018

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

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.

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/dilutio...

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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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