Overview

  • Product name

    Aconitase Assay Kit
    See all Aconitase kits
  • Detection method

    Colorimetric
  • Sample type

    Cell Lysate, Tissue Lysate
  • Assay type

    Enzyme activity
  • Assay time

    0h 40m
  • Product overview

    Aconitase Assay Kit ab83459 is a highly sensitive, simple, direct and HTS-ready colorimetric assay for measuring Aconitase activity in biological samples.


    In the aconitase assay protocol, citrate is converted by aconitase into isocitrate, which is further processed resulting in a product that converts a nearly colorless probe into an intensely colored form with a max absorbance at 450nm.


    Aconitase assay protocol summary:
    - activate aconitase in samples by adding cysteine HCl and (NH4)2Fe(SO4)2
    - add samples and standards to wells
    - add reaction mix and incubate for 30-60 min
    - analyze with a microplate reader

  • Notes

    Aconitase (aconitate hydratase; EC 4.2.1.3) is an iron-sulfur protein containing an [Fe4S4]2+ cluster that catalyzes the stereospecific isomerization of citrate to isocitrate via cis-aconitate in the tricarboxylic acid cycle, a non-redox-active process.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components Identifier 100 tests
    (NH4)2Fe(SO4)2 (Lyophilised) Brown/NM 1 vial
    Assay Buffer WM 1 x 25ml
    Cysteine (Lyophilised) Red 1 vial
    Developer (Lyophilised) Purple 1 vial
    Enzyme Mix Green 1 x 600µl
    Isocitrate Standard (100 mM) Yellow 1 x 100µl
    Substrate (lyophilized) Blue 1 vial
  • Research areas

  • Relevance

    Aconitase (aconitate hydratase; EC 4.2.1.3) is an iron-sulfur protein containing an [Fe4S4]2+ cluster that catalyzes the stereospecific isomerization of citrate to isocitrate via cis-aconitate in the tricarboxylic acid cycle, a non-redox-active process. Tissue contains two aconitases, a mitochondrial (m-) and a cytosolic (c-) aconitase. They are related, but distinctly different enzymes and are coded for on different chromosomes. Loss of aconitase activity in cells or other biological samples treated with prooxidants has been interpreted as a measure of oxidative damage.
  • Cellular localization

    ACO1: Cytoplasmic ACO2: Mitochondrial
  • Alternative names

    • ACO 1
    • ACO 2
    • ACO1
    • ACO2
    • Aconitase 1 soluble
    • Aconitase 2 mitochondrial
    • Aconitate hydratase
    • Aconitate hydratase mitochondrial
    • ACONM
    • ACONS
    • Citrate hydro lyase
    • Cytoplasmic aconitate hydratase
    • EC 4.2.1.3
    • Ferritin repressor protein
    • IRE BP 1
    • IREB1
    • IREBP
    • IREBP1
    • Iron regulatory protein 1
    • Iron responsive element binding protein 1
    • IRP1
    • MGC20605
    • MGC33908
    see all

Images

  • Aconitase measured in mouse tissue lysates showing quantity (mU) per mg of tested sample.

    Protein concentration for samples varied from 1.5 mg/mL to 10 mg/mL. Samples were diluted 1-27 fold.

  • Mitochondrial autophagic profiles in ON vulnerable to secondary degeneration +/- R/NIR-IT. Accumulations of mitochondrial autophagic profiles (red arrows) were seen especially at day 1 after PT (A, B). Occasionally, larger features reminiscent of autophagosomes were observed (A, black arrow) and parts of the nerve appeared highly abnormal (*, B).

  • 30 minute Aconitase sample test using ab83459.
  • Isocitrate standard curve using ab83459.

Protocols

References

This product has been referenced in:

  • Romsang A  et al. Pseudomonas aeruginosa nfuA: Gene regulation and its physiological roles in sustaining growth under stress and anaerobic conditions and maintaining bacterial virulence. PLoS One 13:e0202151 (2018). Read more (PubMed: 30092083) »
  • Langley M  et al. Mito-Apocynin Prevents Mitochondrial Dysfunction, Microglial Activation, Oxidative Damage, and Progressive Neurodegeneration in MitoPark Transgenic Mice. Antioxid Redox Signal 27:1048-1066 (2017). Read more (PubMed: 28375739) »
See all 9 Publications for this product

Customer reviews and Q&As

1-10 of 13 Abreviews or Q&A

Answer



I can confirm that that concentration of the (NH4)2Fe(SO4)2 is approximately 10 mg/ml.

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Answer

The reason for needing to activate the aconitase in the sample is as follows:

The Iron sulphur cluster required for Aconitase activity is inactivated in biological samples when the aconitase is exposed to oxidants/superoxides. The way to measure Aconitase activity in vitro requires reinsertion of ferrous iron into the iron sulphur cluster. This is why you need to use the activation solution, so that the Aconitase in the samples can be tested for activity.

This is a well-studied and documented process reported in literature:
http://www.jbc.org/content/258/18/11098.full.pdf

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Answer

The mU/ml shows activity of Aconitase per unit volume. Another way of expressing the numbers will be if you perform a protein quantitation assay and instead of the ml volume of sample, uses mg of total protein. In this scenario the final numbers can be reported as mU/mg protein. Both of these are equally valid.

To determine protein mass we suggest using a detergent compatible BCA assay for protein quantitation.

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Question
Answer

For ab83459, we have optimized this assay protocol for samples of mammalian origin. However, theoretically this assay should work with samples from multiple origins including bacteria. Since we have not done it ourselves, I do not know if the assay protocol is the most optimized for such samples, so you may have to do some optimization to get the most efficient results.

For these situations when you want to test an antibody or kit in an untested species or application, we have our abTrial program for financially risk-free testing. Information regarding the abTrial program is given through the link below:

https://www.abcam.com/index.html?pageconfig=resource&rid=11998

If you are interested in testing ab83459 in bacteria through the abTrial program, please let me know and I will send you your inactive abTrial discount code.

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Answer

Thank you for contacting us.

I have received word back from the lab regarding your request. Unfortunately the details of the reagents and reactions for these products are proprietary and we cannot share them. They have provided what details of the citrate synthase and PDH system which they are able to. I will attach these to this email.

These products are covered by our Abpromise guarantee. We provide scientific support, replacement or refund should this product not perform as indicated on the datasheet. More information on our Abpromise may be found at the following link:

https://www.abcam.com/index.html?pageconfig=abpromise

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

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Question

Hello!
Thank you for the reply. I mean the aconitase kit ab83459. There is a formula in the section of data analysis.
Good thing about this kit is that a little amount of tissue is necessary for analysis, in my case it would be brain tissue and cell culture.
But another thing that confuse me about this kit is that there is an activation buffer (not preservation one) and the compound with iron, becouse as far as I know according to articles this enzyme might be activated. The point for me is to measure aconitase activtity in the samples considering the sensitivity of this enzyme to ROS, thus it means that aconitase is a sensor of oxydative stress and a tool to estimate the oxydative status of the cell.

Actually the second kit ab109712 seems suitable, but the advisable protein concentration is 5 mg/ml, the problem is that I have less protein concentration (when I have experiments with the particular brain region, there is a really small amount of tissue). how much does the protein concentration affect the assay? I know that there should be some limit. And can the homogenate be used?
The good thing in this kit is the preservation solution instead to activation one.
And speaking about this second kit - the calculations is confusing for me, actually there is no formula, just an example
Example: 12.5 μg bovine heart mitochondria
Rates:
37.3 mOD/min
38.5
39.8
Background: 0.4 mOD/min,
Corrected rate: 38.1±1.3 mOD/min
= 17.3 μM/min
0.25 mL/well = 4.3 nmoles/min
12.5 μg /well = 0.35 μmoles/min/mg mitochondria
Extinction coefficient = 2.2 OD mM-1/well*

So, it means to take 3 OD of the sample from linear part of the kinetic curve, calculate the average rate, then minus background, then divide by sample volume and Extinction coefficient, is it wright?

Thank you in advance,
Best wished,

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Answer

Thank you for your interest in our products.

I have been discussing your enquiry with my colleagues in the Lab and would like to share the following information:

Regarding ab109712: Aconitase Enzyme Activity Microplate Assay Kit

I can confirm that a homogenate can be used but it needs to be very concentrated (no less than 2mg/mL). There is no need to extract mitochondria so long as the homogenate is concentrated. You must ensure that the tissue is NOT homogenized in the preservation buffer (which is only required for cells). We have tested in the past Rat liver tissue homogenized in 10mM Tris pH 7.4 + 250mM Sucrose buffer +0.2mM EDTA (you may add 2mM sodium citrate and/or 2.5mM MnSO4 during the homogenization procedure to maintain a stable aconitase enzyme). From personal experiencewe have found that, at least in Rat liver homogenate stored at -80C at a concentration of 25mg/mL and solubilized at 5mg/mL, Citrate and MnSO4 do not improve on the aconitase signal obtained with the simple sucrose buffer. However these additives may be useful in other tissues/homogenates stored at lower concentration.

The protein concentration is important for optimal performance. One main reason is that for optimal signal (at least for rat liver homogenate) samples should be loaded at 100ug/50uL (2mg/mL) and therefore a calibration curve should be set to go above and below is concentration. There is also the issue of protein to detergent ratio during the solubilization of the homogenate.

For the calculations =

experiments should be run in triplicates on kinetic mode reading milliODs and not ODs.
Always run a background (no sample)
Always run a calibration curve. For Rat liver homogenate for example the limit of detection has been found at 100ug/mL (50uL loading prior to the addition of 200uL of activity buffer) and the range has been found between 7mg/mL to 0.1mg/mL (again in the 50uL loading). This would change from tissue to tissue.
Data will come out of the instrument in mOD/min, this can be divided by the extinction coefficient to obtain the molar equivalency.



Regarding ab83459: Aconitase Assay Kit

Yes, this kit comes with an activation solution components instead of preservation ones. Aconitase preservation solution will maintain aconitase activity during sample preparation whereas an inactivated aconitase may be activated in vitro by the addition of iron and cysteine.

Exposure of aconitase to oxidants renders the enzyme inactive and loss of aconitase activity in cells or in biological samples treated with pro-oxidants has been interpreted as a measure of oxidative damage.

It is this aconitase which will be activated for measuring with this kit.

Hope this information has been helpful for you.

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Answer

Thank you for contacting us. You may be interested in the following kits:

Citrate Synthase: ab119692
Aconitase: ab109712 or ab83459
CitrateLyase: wedo not currently have a kit available, but weyou may be interested in ourprimary antibodies ab40793 and ab61762
NAD/NADH: ab65348 (we also havea kit forNADH Dehydrogenase: ab124535)
Isocitrate Dehydrogenase: ab102528
Isocitrate Lyase: no kits or antibodies currently available

I hope this helps, please let me know if you need any additional information or assistance.

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Question
Answer

Thank you for contacting us.
I had chat with the product manager of this assay kit and would like to confirm that we are happy to provide you 100% Abreview discount code for this product. The discount code will be, worth the price you pay for ab83459. In brief, the procedure to avail the discount is as follows.
- Confirm to me if you are interested in the discount offer, I will then create a discount code for you.
- Buy the kit ab83459 at full price
- Send me all the experimental details and results that we can publish online; any sensitive information will be omitted
- I will process the Abreview and will validate the discount code.
- The valid discount code can be used to get a free kit or can be used to save similar value from any other purchase.
I am hoping that this kit will be a perfect fit with your samples. Once you try the first one and happy with the results, I will then send you a discounted quote for purchase of 8-10 kits.
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 contacting us.

I have been in touch with the labs regarding use of ab109712 &ab83459 for duodenumtissue because of its iron aborbence.

In regards to ab109712:Although we have not tested in this tissue type, there have been studies on aconitase and duodenal iron – one paper shows no effect.http://www.ncbi.nlm.nih.gov/pubmed/8541321

In other studies:http://www.ncbi.nlm.nih.gov/pubmed?term=iron%20dudenum%20aconitase Ppresumably the iron is sequestered after absorption preventing significant damage.



In regards to ab83459:We do not think this will be a problem, but we have not tested duodenum.



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

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Question
Answer

Thank you for your patient. There are two kits in our catalogue targeting Aconitase: ab83459 and ab109712. Both are compatible with frozen tissues, as long as they are frozen appropriately preserving the enzymatic activity. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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1-10 of 13 Abreviews or Q&A

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