Overview

  • Product name
    Aconitase Enzyme Activity Microplate Assay Kit
    See all Aconitase kits
  • Detection method
    Colorimetric
  • Sample type
    Cell culture extracts, Tissue
  • Assay type
    Enzyme activity
  • Assay time
    2h 00m
  • Assay duration
    Multiple steps standard assay
  • Product overview

    ab109712 is a simple, reproducible, and sensitive tool for assaying aconitase from tissue homogenates or cell lysates. Unlike other assays this is not a coupled reaction and therefore only aconitase activity is required and measured. Aconitase catalyzes an equilibrium between aconitate, cis-aconitate and iso-citrate. These reactions are monitored by measuring the increase in absorbance at 240 nm associated with the formation of the cis-aconitate which has an extinction coefficient of 2.2 OD/mM per well. Therefore the rate of cis-aconitate production is proportional to aconitase activity.

     

    Aconitase preservation solution, assay buffer, reagents and an essential UV microplate are provided for this measurement. The entire assay can be completed within 2 hours.

    Note – mitochondrial and cytoplasmic aconitase activities are indistinguishable. Therefore, to measure the mitochondrial activity only, first isolate mitochondria, or for both activities fractionate the cells into cytoplasmic and mitochondrial.

  • Notes

    Aconitase (aconitate hydratase; EC 4.2.1.3) is an iron-sulfur protein that catalyzes the reversible inter-conversion of citrate and isocitrate, via a cis-aconitate intermediate, in both the TCA and glyoxylate cycles. The enzyme contains a [4Fe-4S] cluster which interacts directly with the substrates. In eukaryotes there are both mitochondrial and cytosolic forms of the enzyme. The mitochondrial form functions not only in the TCA cycle, but also to stabilize mtDNA thereby influencing mitochondrial gene expression. The cytosolic form can function as an aconitase as well as an iron regulatory protein.

    The active form of the enzyme is inhibited by citrate analogs, and fluoracetate. Other inhibitors include oxidative stress agents such as peroxynitrite, hydrogen peroxide and superoxide, which inactivate the enzyme by changing the [4Fe-4S] to a [3Fe-4S] cluster. Aconitase is considered a good marker of mitochondrial and cellular oxidative stress. This change in mitochondrial aconitase can lead to a decreased energy production, whereas in cytosolic aconitase it triggers binding of the enzyme to mRNA iron response elements resulting in increased expression of iron uptake proteins and decreased transcription of iron sequestering protein.

    A hydroxyl scavenging solution (Aconitase preservation solution) is supplied with this kit to maintain aconitase activity during sample preparation. An inactivated [3Fe-4FS] aconitase may be activated in vitro by the addition of iron and cysteine.

  • Platform
    Microplate

Properties

  • Storage instructions
    Store at +4°C. Please refer to protocols.
  • Components 96 tests
    10X Detergent 1 x 1ml
    96-well UV microplate 1 unit
    Aconitase Preservation Solution 1 x 20ml
    Buffer 1 x 50ml
    Isocitrate (25X) 1 x 800µl
    Manganese (100X) 1 x 200µl
  • 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

  • Mitochondria were isolated from HL-1 cells following starvation protocol and approximately 50μg of mitochondrial preparation were placed in each microplate well. Equal amounts of the substrate isocitrate were added to all wells and the absorbance at 240nm was recorded for 30 minutes. The catalytic activity was measured by the rate of formation of cis-aconitate as detected by the increase in absorbance.

  • Figure 1. Bovine heart mitochondria were treated with increasing concentrations of hydrogen peroxide and peroxynitrite to generate aconitase activity IC50's for these oxidative stress agents.
  • Figure 2. Using cellular fractionation kit ab109719, whole cells were separated into cytoplasmic, mitochondrial and nuclear fractions.

Protocols

References

This product has been referenced in:
  • Kauppila JHK  et al. Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice. Nucleic Acids Res N/A:N/A (2018). WB . Read more (PubMed: 29860357) »
  • Vannocci T  et al. Adding a temporal dimension to the study of Friedreich's ataxia: the effect of frataxin overexpression in a human cell model. Dis Model Mech 11:N/A (2018). Read more (PubMed: 29794127) »
See all 9 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Answer

The kit should be OK. Please bring all the components to room temperature before preforming the assay.

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Answer

Thank you for contacting us.


There is manganese in the buffer (final 0.5 mM) which was developed and shown to maintain the aconitase activity of a sample on ice over a long period of time.This was true for whole cell lysates and isolated mitochondria. I agree, I found two reports one in 2007 indicates that manganese inhibits m-aconitase, this would correspond to approximately 10% reduction in activity at 0.5mM using an isocitrate dehydrogenase coupled assay, we did not see this in the direct aconitase assay we use. Fortunately the manganese is a supplement, if you have any concerns it can be left out of the assay. In this case it would be important to limit the sample time on ice.

The preservation solution contains a variety of antioxidants to preserve the aconitase activity over time (hours) on ice. As with the manganese it can be omitted if desired.


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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Answer

Thank you for contacting us and your interest in our products.

The Aconitase Enzyme Activity Microplate Assay Kit (ab109712) should be compatible with bacterial samples, however as we have not performed this assay ourselves, we would not be able to guarantee the performance of the kit and it is likely to require some optimisation. Effective bacterial lysis in order to release the cytoplasm without destroying the aconitase activity would be crucial and unfortunately, this is not something we would be able to advise further on.

I hope this information has been of some help. If I can be of further assistance, please do not hesitate to let me know.

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Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.


I look forward to receiving your reply.

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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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Answer

Thank you for your call yesterday and for your patience while I've been in touch with the lab regarding your enquiry.

I've heard back from the scientists in the lab, and they recommend the following:

"We typically do BCA assays on extracts solublized with the detergent. However we don’t measure neat extract by BCA, instead we dilute 1:10 to 1:40 in 0.25% SDS before loading on the BCA plate. The standard curve (BSA) is also diluted in 0.25% SDS. After calculating protein concentration,you should remember to multiply by the dilution factor if the instrument cannot be set up to do it automatically.

The buffer in the activity kit contains 0.1% BSA, so this will certainly increase the background.The protein in samples must be measured before they are diluted in the preservation buffer (which contains BSA) and the preservation buffer or buffer in the kit should not be used for BCA assay. The suggestion is to dilute a portion of the sample extract not in the kit’s buffer but in 0.25% SDS. SDS will fully denature the protein extracted with the kit’s detergent (which solubilizes enzymes into their native form). Cysteines, tyrosines and tryptophans of fully denatured proteins will react faster on the BCA assay.The samples could also be diluted in the detergent provided for the BCA assay. However the reaction will be slower.Cultured cell lysates react extremely fast on the BCA assay. We recommend to allow the assay to fully develop at 37C for 2 hours before reading, otherwise protein quantity will be overestimated. We think this fast reaction may be due to glutathione (free cys) reaction."

I hope this information will be useful, but if you have any further questions or if there is anything else that we can do for you, please let me know and I'll be happy to help.

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Answer

Thank you for contacting us.

The function of aconitase preservation solution is to stabilize aconitase activity for more than 3 hours on ice upon addition into your sample.


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

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