MitoSciences (MS745)

Aconitase Enzyme Activity Microplate Assay Kit (ab109712)

Overview

  • Product nameAconitase Enzyme Activity Microplate Assay Kit
    See all Aconitase kits
  • Detection methodColorimetric
  • Tests
    1 x 96 well plate
  • Sample type
    Cell culture extracts, Tissue
  • Assay typeEnzyme activity
  • Assay time
    2h 00m
  • Assay durationMultiple 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.

  • Tested applicationsFunctional Studiesmore details
  • PlatformMicroplate

Properties

  • RelevanceAconitase (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 localizationACO1: 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

Applications

Our Abpromise guarantee covers the use of ab109712 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Functional Studies Use at an assay dependent dilution.

Aconitase Enzyme Activity Microplate Assay Kit images

  • Figure 2. Using cellular fractionation kit ab109719, whole cells were separated into cytoplasmic, mitochondrial and nuclear fractions.
  • 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.

Protocols

References for Aconitase Enzyme Activity Microplate Assay Kit (ab109712)

This product has been referenced in:
  • Cheng X  et al. Ablation of promyelocytic leukemia protein (PML) re-patterns energy balance and protects mice from obesity induced by a Western diet. J Biol Chem 288:29746-59 (2013). Mouse . Read more (PubMed: 23986437) »
  • Jiang H  et al. Hepatocytes Determine the Hypoxic Microenvironment and Radiosensitivity of Colorectal Cancer Cells Through Production of Nitric Oxide That Targets Mitochondrial Respiration. Int J Radiat Oncol Biol Phys N/A:N/A (2012). Read more (PubMed: 22975619) »

See all 4 Publications for this product

Product Wall

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

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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 isolate...

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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 ab...

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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 at...

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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 Read More

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 ant...

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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...

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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 d...

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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, the...

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Thank you very much for your phone call.

The kit ab109712 utilizes an Aconitase specific antibody which is coated on the ELISA plate and tested using human cell culture and tissue culture extracts. We unfortunately haven't tested this kit wit...

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